Dissertations / Theses: 'RNA exosome'

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Giacometti, Simone. "CBC bound proteins and RNA fate." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTS028.

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Le complexe de liaison de la coiffe des ARN (CBC) joue un rôle essentiel dans leur maturation et déclenche une variété de réactions biochimiques, via son interaction avec différents partenaires. Deux complexes, CBC-ARS2-PHAX (CBCAP), et CBC-ARS2-ZC3H18-NEXT (CBCN), ont récemment été montré comme important pour cibler les ARN vers l'export (CBCAP) ou la dégradation (CBCN). Cependant, les mécanismes par lesquels la sélection se fait pour l'une voie ou l'autre reste mystérieuse. Ainsi, une question majeure qui reste à résoudre est de savoir quand et comment ces complexes sont recrutés sur les ARN. Dans ce travail, j'ai utilisé la procédure du iCLIP (Cross-Linking and Immuno-Precipitation), afin d'identifier les cibles de ces complexes sur l'ensemble du transcriptome humain. J'ai réalisé un iCLIP sur cinq composants de CBCAP et CBCN, et j'ai comparé les résultats à ceux obtenus avec RBM7, un composant de NEXT précédemment étudié par iCLIP. Mes résultats indiquent que: (i) CBP20, ARS2, PHAX et ZC3H18 se lient près de la coiffe des ARN, tandis que RBM7 et MTR4 se lient partout; (ii) CBP20, ARS2, PHAX et ZC3H18 s'associent à un large ensemble d'ARN transcrits par l'ARN polymérase II et montrent une faible sélectivité; (iii) la liaison de ces protéines varie avec l'état de maturation des ARN, avec le CBC enrichi sur les ARN matures, tout comme ARS2/PHAX/ZC3H18 et MTR4 (bien que dans une moindre mesure), tandis que RBM7 est préférentiellement lié sur les pre-mRNAs non épissés; (iv) une liaison différentielle de RBM7 et MTR4 sur les ARN, avec RBM7 enrichi sur les introns et les PROMPTs, et MTR4 plus présent sur les ARN mature. Bien que des expériences additionnelles soient requises, nous proposons que le CBCAP et le CBCN se lient à un même ensemble d'ARN, ce qui indique à la fois une compétition entre ZC3H18 et PHAX pour la liaison à ces ARN, et l'absence de voies de routage bien déterminées qui ciblerait les ARN vers l'une ou l'autre de ces protéines. Le devenir des ARN pourrait ainsi être déterminé par d'autres caractéristiques des ARN, ou encore par des protéines additionnelles. Ces facteurs pourraient s'allier aux protéines liées à la coiffe afin de favoriser la formation du CBCAP ou du CBCN. Dans le but d’identifier des facteurs additionnels, j'ai réalisé un screen d'interaction par spectrométrie de masse après purification de ARS2 ou CBP80. Ceci a été fait dans des conditions natives ou après un cross-link des complexes à la formaldéhyde, afin de stabiliser les interactions transitoires. Ceci a permis d'identifier de nouveaux partenaires de ARS2 et de CBP80, dont la majorité sont impliqués dans l'épissage des ARN. Des expériences additionnelles seront nécessaires pour valider ces interactions
The cap-binding complex (CBC) plays a pivotal role in post-transcriptional processing events and orchestrates a variety of metabolic pathways, through association with different interaction partners. Two CBC sub-complexes, the CBC-ARS2-PHAX (CBCAP) and the CBC-nuclear exosome targeting (NEXT) complex (CBCN), were recently shown to target capped RNA either toward export or degradation, but the mechanisms by which they can discriminate between different RNA families and route them toward different metabolic pathways still remain unclear. A major question to be answered is how and when the different CBC subcomplexes are recruited to the RNP. Here, we used an individual nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) approach to identify the transcriptome-wide targets for 5 different components of the CBCAP and CBCN complexes, and compared results to the previously analysed NEXT-component RBM7. We report that: (i) CBP20, ARS2, PHAX and ZC3H18 bind close to the cap, while RBM7 and MTR4 bind throughout the mRNA body; (ii) CBP20, ARS2, PHAX and ZC3H18 associate with a broad set of RNA polymerase II (PolII)-derived RNAs and have only mild species preferences; (iii) binding varies with the RNA maturation stage, with the CBC being highly enriched on mature mRNA, ARS2/PHAX/ZC3H18/MTR4 less so, and RMB7 preferentially bound to pre-mRNAs; (iv) MTR4 and RBM7 show different specificities, with RBM7 being highly enriched on introns and promoter upstream transcripts (PROMPTs), while MTR4 is additionally present on mature RNAs. Although more experimental work is needed to fully support our model, we propose that CBCAP and CBCN bind overlapping sets of RNAs, indicating a competition between the proteins ZC3H18 and PHAX, and the lack of a strict RNA sorting mechanism. RNA fate may therefore be determined by additional RNA features and/or by other RNA-binding proteins, which may synergize with the cap and drive the formation of one specific CBC subcomplex instead of another. In an attempt to identify yet unknown factors that may interact with cap-bound CBCAP and CBCN, we performed a protein interaction screen leveraging affinity capture-mass spectrometry (ACMS), using ARS2 and CBP80 as bait proteins. As a complementary approach, we also employed a formaldehyde-based chemical cross-linking strategy, aimed at stabilizing weak/transient interactions. Although we failed to detect any transient interactions involving the CBC, we identified several potential CBC80 and ARS2 interactors, the majority of which are involved in pre-mRNA splicing. Additional quantitative experiments are required to validate our ACMS results and confirm the existence of such protein interactions in vivo

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2

Paiva, Germano Alves. "Estudo do papel de Rrp43p na montagem e estabilização do complexo do exossomo em Saccharomyces cerevisiae." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-09052013-105201/.

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O exossomo é um complexo constituído por até 11 subunidades (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p, Mtr3p) que possui atividade exorribonucleolítica 3`→5` e está envolvido no processamento e degradação de vários tipos de RNAs na célula eucariótica. O complexo tem sido estudado em diversos organismos, como leveduras, insetos, plantas, humanos e também em várias espécies de archaea. Apesar da conservação da estrutura do exossomo ao longo da evolução e de oito subunidades do exossomo eucariótico apresentarem domínios de RNase, apenas duas proteínas, Rrp6p e Rrp44p têm atividade catalítica. A despeito da importância do exossomo para a célula, ainda não está claro o papel de cada subunidade na atividade do complexo. Neste trabalho foram utilizados mutantes da subunidade Rrp43p a fim de avaliar como mutações pontuais nesta subunidade afetam a montagem e estabilização do complexo do exossomo de Saccharomyces cerevisiae. Ensaios de purificação do exossomo com TAP-Rrp43p revelaram que os mutantes co-purificam Mtr3p e Rrp44p menos eficientemente. Além disso, os mutantes também apresentam atividade exorribonucleolítica 3`→5` reduzida, indicando que o defeito na montagem do complexo pode afetar a sua atividade enzimática.
The exosome is a protein complex comprised of up to eleven subunits (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p and Mtr3p) that has 3`→5` exoribonucleolytic activity and is involved in degradation and processing pathways of several kinds of RNA in eukaryotes. This complex has also been identified in several organisms, such as yeast, insects, plants, humans and also many species of archaea. Despite the overall structure conservation of the complex throughout evolution and eight of the eukaryotic exosome subunits displaying RNase domains, only two proteins, Rrp6p and Rrp44p have catalytic activity. Although the exosome has been shown to be involved in many different aspects of RNA metabolism, the role that each subunit plays in the activity of the complex has not yet been determined. In this work we used of TAP-purified exosome complexes to study the effect of Rrp43p mutations on the assembly and stabilization of the complex in Saccharomyces cerevisiae. Co-immunoprecipitation assays revealed that Rrp43p mutants co-purify Mtr3p and Rrp44p subunits less efficiently. Besides, Rrp43p mutants also present decreased activity, indicating that an assembly defect may affect its enzymatic activity

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3

Kiss, Daniel L. "The Exozyme Model: A New Paradigm of Exosome Subunit Activity Revealed by Diverse and Distinct Substrate Specificities of Exosome Subunits In Vivo." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1263237977.

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4

Wlotzka, Wiebke. "RNA-protein crosslinking identifies novel targets for the nuclear RNA surveillance machinery." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5047.

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The RNA binding proteins Nrd1 and Nab3 function in transcription termination by RNA Pol II, acting via interactions with the CTD of the largest polymerase subunit, particularly on snRNA and snoRNA genes. They also participate in nuclear RNA surveillance and ncRNA degradation, functioning together with the exosome and the Trf-Air-Mtr4 polyadenylation (TRAMP) complexes. To better understand the signals for surveillance and ncRNA degradation, I applied an RNA-protein crosslinking approach in combination with Solexa sequencing. This approach identified in vivo binding sites for Nrd1, Nab3 and Trf4. Several million sequences were recovered and mapped to the yeast genome. This identified three classes of substrates: 1) Expected targets, including snRNAs, snoRNAs and characterized short ncRNAs. 2) Unknown but anticipated substrates, including several hundred previously uncharacterized ncRNAs that lie antisense to protein coding genes (asRNAs). 3) Unexpected targets, including many Pol III transcribed precursor RNAs. Bioinformatics analyses of the high-throughput sequencing data revealed that known binding motifs for Nrd1 and Nab3 were frequently recovered. Many recovered RNAs contained non-templated oligo(A) tails with an average of 2-5 nt length. This clearly distinguishes targets for surveillance machinery from polyadenylated mRNAs that get stabilized by polyadenylation (A70-90 in yeast). For a few selected, predicted asRNAs I was able to validate the crosslinking data by demonstrating that corresponding long RNAs are both detectable and increased by loss of Nrd1, Nab3, Trf4 or the exosome component Rrp6. Interestingly, loss of Nrd1 or Nab3 led to transcriptional read through on long asRNA transcripts. In addition, I have identified pre-TLC1 (telomerase RNA) as a target for the surveillance machinery. Processing of this long ncRNA was only poorly characterized in yeast but I could demonstrate that its transcription termination and maturation is mainly dependent on actions of the Nrd1-Nab3-Sen1, TRAMP4 and exosome complexes. It was previously reported that Nrd1-Nab3 acts only on short RNAs, due to the association with Ser5 phosphorylated CTD. My findings suggest that action of Nrd1- Nab3 is not exclusively on Ser5 phosphorylated form of the CTD. Unexpectedly the Pol II associated factors Nrd1 and Nab3 bound Pol III precursor transcripts. Surveillance of Pol III transcripts was dependent on Nrd1 and Nab3 since depletion of Nrd1 or Nab3 led to accumulation of pre-tRNAs. In addition, I could demonstrate that pre-RNase P RNA is oligoadenylated in vivo, which was dependent on Nrd1, Nab3 and Trf4. Together, my findings suggest a revised model of nuclear RNA surveillance in which Nrd1-Nab3 not only acts in co-transcriptional RNA recognition on Pol II transcripts but also post-transcriptionally on Pol III RNAs. The TRAMP complex is recruited to the defective RNA by the Nrd1-Nab3 complex, which remains associated with the RNA through the process of polyadenylation, until the exosome degrades the aberrant transcript.

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5

Hessle, Viktoria. "Characterization of RNA exosome in Insect Cells : Role in mRNA Surveillance." Doctoral thesis, Stockholms universitet, Institutionen för molekylärbiologi och funktionsgenomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-52127.

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The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects.
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

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Witharana, Chamindri [Verfasser]. "The heterogeneity of the RNA degradation exosome in Sulfolobus solfataricus / Chamindri Witharana." Gießen : Universitätsbibliothek, 2013. http://d-nb.info/1065395418/34.

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Feigenbutz, Monika U. "Role of the exosome co-factor Rrp47 in RNA processing and surveillance." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/5285/.

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RNA surveillance by the exosome complex is remarkably conserved from yeast to humans and best studied in baker's yeast Saccharomyces cerevisiae. The multi-subunit RNA exosome is involved in the processing, maturation, quality control and general turnover of RNAs, as well as the degradation of harmful, aberrant or unwanted transcripts. To execute its distinct cytoplasmic and nuclear functions, the exosome requires compartment-specific co-factors like Rrp47, a protein directly associated with the nuclear exosome exoribonuclease Rrp6. The aim of this study was to investigate the role of Rrp47 in exosome-mediated processes based on the model that Rrp47 is an RNA binding protein that helps direct Rrp6 to its substrates. Mutational analysis of Rrp47 revealed that the Sas10 domain which Rrp47 shares with other proteins involved in RNA processing is critical for Rrp6 binding and for all in vivo Rrp47 functions. However, the less conserved C-terminus of Rrp47 functions in the final maturation of snoRNAs, and both C- and N-terminus cooperate in RNA binding in vitro. Protein and mRNA expression analyses demonstrate that the proteins critically influence each other's stability and expression levels whereby Rrp47 expression is drastically reduced when Rrp6 is absent. Studies into the assembly of Rrp47-Rrp6 suggest that the proteins are imported into the nucleus separately where Rrp47 is degraded if Rrp6 is not available for interaction. Rrp47 has less pronounced effects on Rrp6 stability and expression, yet defects in the processing of Nrd1 terminated transcripts were alleviated by Rrp6 overexpression in cells lacking Rrp47. Specifically, growth was restored by overexpressing Rrp6 in an otherwise synthetic lethal rex1? rrp47? strain, suggesting that Rrp47 is critical for maintaining adequate Rrp6 levels. Taken together this study has given crucial new insights into domains required for Rrp47 function, as well as assembly and interdependency of Rrp47 and its associated exonuclease Rrp6.

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Rege, Mayuri. "RNA Exosome & Chromatin: The Yin & Yang of Transcription: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/812.

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Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood. We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome. Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis.

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Jackson, Ryan N. "Structural and Functional Characterization of the Essential RNA Helicase Mtr4." DigitalCommons@USU, 2012. https://digitalcommons.usu.edu/etd/1414.

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The essential protein Mtr4 is a conserved Ski2-like RNA helicase that maintains the integrity of nuclear RNA by promoting the 3' end decay of a wide variety of RNA substrates. Mtr4 activates the multi-protein exosome in RNA processing, surveillance, and turnover pathways by unwinding secondary structure and/or displacing associated proteins from RNA substrates. While Mtr4 may be able to promote decay independently, it is often associated with large multi-protein assemblies. Specifically, Mtr4 is the largest member of the TRAMP (Trf4/Air2/Mtr4 polyadenylation) complex which targets a plethora of RNA substrates for degradation by appending them with small (~5nt) poly(A) tails via the polymerase activity of Trf4. Mtr4 preferentially binds and unwinds RNAs with short poly(A) tails. Notably, the mechanism by which Mtr4 recognizes the length and identity of the RNA 3' end is coupled to the modulation of poly(A) polymerase activity of Trf4. The lack of structural data for Mtr4 and associated complexes severely limits the understanding of Mtr4 function. Particularly, it is unclear how Mtr4 senses RNA features, acts on RNA substrates, delivers RNA substrates to the exosome, and assembles into larger protein complexes. Presented here is the x-ray crystal structure of Mtr4 combined with detailed structural and biochemical analysis of the enzyme. The structure reveals that Mtr4 contains a four domain helicase core that is conserved in other RNA helicases and a unique arch-like RNA binding domain that is required for the in vivo processing of 5.8S rRNA. Furthermore, kinetic and in vivo analysis of conserved residues implicated in the poly(A) sensing mechanism demonstrates that ratchet helix residues regulate RNA unwinding and impact RNA sequence specificity. A comparison of the apo Mtr4 structure with the RNA/ADP bound structure (determined elsewhere) provides a view of the range of motion that individual domains of Mtr4 adopt upon substrate binding as well as the possible conformations that occur during RNA translocation. These studies provide an important framework for understanding the fundamental role of Mtr4 in exosome-mediated RNA decay, and more broadly describe common themes in architecture and function of the Ski2-like helicase family.

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Kong, Ka-yiu, and 江家耀. "Characterization of the roles of yeast nuclear exosome cofactor TRAMP complex in pre-mRNA splicing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193522.

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In budding yeast, the Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex recognizes unwanted RNA transcripts in the nucleus and then targets them to the nuclear exosome for rapid degradation, constituting an important pathway of nuclear RNA quality control. Each pre-mRNA splicing event unavoidably generates a RNA side-product that should be recognized by TRAMP and then removed by the nuclear exosome to prevent the potentially harmful sequestration of splicing factors and/or ribonucleotides. While successful pre-mRNA splicing inevitably produces a spliced-out intron, errors in pre-mRNA splicing lead to the emergence of either an abnormal splicing intermediate, or a splicing-incompetent pre-mRNA that cannot be properly spliced. However, it remains unclear how and when these RNA side-products of pre-mRNA splicing are recognized by TRAMP. In this study, chromatin immunoprecipitation (ChIP) was applied to demonstrate that both TRAMP and the nuclear exosome component Rrp6p are cotranscriptionally recruited to nascent RNA transcripts, particularly to intronic sequences, indicating that splicing side-products are recognized by TRAMP and committed to subsequent nuclear-exosome-mediated degradation in a cotranscriptional manner. Deletion of TRF4, of both AIR1 and AIR2, or of RRP6, resulted in accumulation of unspliced pre-mRNAs. Surprisingly, while such pre-mRNAs accumulated in rrp6 cells owing to defects in pre-mRNA degradation, the same phenotype in trf4 and air1air2 cells involved splicing defects, demonstrating that only TRAMP, but not the nuclear exosome, contributes to optimal pre-mRNA splicing. Consistent with a direct stimulatory role for TRAMP in pre-mRNA splicing, negative genetic interactions and physical interactions between Trf4p and several splicing factors were observed, and that Trf4p was further shown to be required for optimal recruitment of the splicing factor Msl5p. The direct facilitation of pre-mRNA splicing by TRAMP may act as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP to nascent intron-containing transcripts before or during pre-mRNA splicing, such that the subsequently generated spliced-out introns, abnormal splicing intermediates, or splicing-incompetent pre-mRNAs can be recognized immediately by TRAMP, and then targeted to the nuclear exosome for prompt degradation before their potentially harmful accumulation. Since most TRAMP and nuclear exosome components found in budding yeast also contain functional human homologs, this work provides important insights into how splicing side-products are rapidly degraded by the nuclear RNA quality control system in human cells, which have a much higher frequency of introns within their genome, and certainly require a much more efficient pathway for the removal of an increased amount of splicing side-products due to the greater number of splicing events.
published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy

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11

Granato, Daniela Campos. "Caracterização da função da proteína Nop53p de Saccharomyces cerevisiae." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-31012008-081416/.

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Em eucariotos, o processamento de pré-rRNA depende de vários fatores como endonucleases, exonucleases, RNA helicases, enzimas modificadoras de rRNA e componentes de snoRNPs. Com o objetivo de caracterizar novas proteínas envolvidas no processamento de pré-rRNA, foi identificada a proteína Nop53p interagindo com a proteína nucleolar Nop17p a partir de uma varredura da biblioteca de cDNAs de Saccharomyces cerevisiae. A cepa condicional contendo a seqüência da ORF NOP53 sob controle do promotor de galactose não cresce em meio contendo glicose, indicando que Nop53p seja uma proteína essencial para a viabilidade celular. Os resultados deste trabalho demonstram que Nop53p está envolvida nas etapas iniciais de clivagem do pré-rRNA, assim como nas clivagens responsáveis pela formação dos rRNAs maduros 5.8S e 25S. Análise mais detalhada do processamento de pré-RNA por Northern blot e \"pulse-chase labeling\", revelou também que Nop53p afeta principalmente o processamento do rRNA intermediário 27S, que origina os rRNAs maduros 5.8S e 25S. Nop53p participa do processamento desses rRNAs afetando a poliadenilação dos precursores dos rRNAs 5.8S e 25S. Experimentos de co-imunoprecipitação de RNA com a proteína de fusão ProtA-Nop53p confirmaram o envolvimento de Nop53p no processamento do 27S rRNA, indicando que essa proteína possa ligar RNA diretamente. A capacidade de Nop53p de ligar RNA foi confirmada através de testes in vitro, enquanto que ensaios de co-imunoprecipitação de cromatina revelaram que Nop53p liga-se ao rRNA 5.8S durante a transcrição. Nop53p regula a função do exossomo através da sua interação direta com a subunidade exclusivamente nuclear deste complexo, Rrp6p.
In eukaryotes, the rRNA processing depends on several factors, such as, endonucleases, exonucleases, RNA helicases, rRNA modifying enzymes and components of the snoRNPs. With the purpose of characterizing new proteins involved in pre-rRNA processing, Nop53p was identified interacting with the nucleolar protein Nop17p in a two hybrid assay. The conditional yeast strain containing the sequence of the ORF NOP53 under the control of the galactose promoter cannot grow in medium containing glucose, indicating that the protein is essential for cell viability. The results of this work demonstrate that Nop53p is involved in the initial steps of pre-rRNA processing and in the cleavages responsible for the formation of the mature rRNAs 5.8S and 25S. A more detailed analysis of the pre-rRNA processing, by Northern blot and pulse-chase labeling, revealed that Nop53p affects the processing of the 27S precursor, that originates the rRNAs 5.8S and 25S. Nop53p participates in the processing of these RNAs by affecting the polyadenylation of the precursors of the rRNAs 5.8S and 25S. RNA co-imunoprecipitation assays with the fusion protein A-Nop53p confirmed the involvement of Nop53p in the processing of the 27S pre-rRNA, indicating that the protein may interact directly with the RNA. The capacity of Nop53p to bind RNA was confirmed by in vitro assays, while chromatin imunoprecipitation assays demonstrated that Nop53p binds the 5.8S rRNA co- transcriptionally. Nop53p regulates the function of the exosome by interacting directly with the exclusively nuclear subunit of the complex, Rrp6p.

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Cvetković, Miloš [Verfasser], and Remco [Akademischer Betreuer] Sprangers. "NMR Studies of RNA Degradation by the Archaeal Exosome Complex / Miloš Cvetković ; Betreuer: Remco Sprangers." Tübingen : Universitätsbibliothek Tübingen, 2017. http://d-nb.info/1165577585/34.

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Luz, Juliana Silva da. "Análise estrutural e funcional de cofatores do exossomo em Saccharomyces cerevisiae e Pyrococcus." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-29092006-124545/.

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A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea.
The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.

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Cordiner, Ross Andrew Alex. "The cellular functions of the microprocessor complex." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25877.

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DGCR8 (DiGeorge critical region 8) protein constitutes part of the Microprocessor complex together with Drosha, and is involved in the nuclear phase of microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin. Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP of DGCR8 protein revealed that this complex also binds and regulates the stability of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA) transcripts, since the stability of these transcripts is dependent on DGCR8, but independent of Drosha. This raises the interesting possibility that there could be alternative DGCR8 complex/es using different nucleases to process a variety of cellular RNAs. We performed mass spectrometry experiments and revealed that DGCR8 copurifies with subunits of the nucleolar exosome, which contains the exonuclease RRP6. We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the mature snoRNAs tested within this study. Interestingly, we also show that DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere phenotype. In order to identify the RNA targets of the DGCR8/Exosome complex on a global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6 identified common substrates; accordingly snoRNAs were the most represented. In addition, we identified the cellular RNA targets of the RRP6 associated human exosome. The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions that are highly dynamic and transient and also highlighted the role of RRP6-mediated trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the RNA-binding capacity of the RRP6-associated exosome. In sum, we identified a novel function for DGCR8, acting as an adaptor to recruit the exosome to structured RNAs and induce their degradation. Moreover, we have identified DGCR8-depenedent substrates of the exosome and have demonstrated the requirement of RRP6 for 3’ processing of ncRNAs.

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15

Santos, Márcia Cristina Teixeira dos. "Caracterização funcional da proteína Nop8p de Saccharomyces cerevisiae." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-28052012-100927/.

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A proteína nucleolar Nop8p de levedura foi identificada inicialmente através de sua interação com Nip7p e está envolvida na formação da subunidade ribossomal 60S. A depleção de Nop8p em células de levedura leva à degradação prematura dos rRNAs, porém o mecanismo bioquímico responsável por este fenótipo ainda não é conhecido. Neste trabalho, mostramos que a interação de Nop8p com o rRNA 5.8S se dá através de sua região amino-terminal, enquanto que a porção carboxi-terminal é responsável pela interação com Nip7p e complementa parcialmente o defeito no crescimento observado na cepa mutante condicional Δnop8/GAL::NOP8. Além disso, Nop8p media a associação de Nip7p com as partículas pré-ribossomais. Nop8p também interage com a subunidade Rrp6p do exossomo e inibe a atividade do complexo in vitro, sugerindo que a diminuição dos níveis da subunidade ribosomal 60S detectada após a depleção de Nop8p pode ser resultado da degradação dos pré-rRNAs pelo exossomo. Estes resultados indicam que Nop8p pode regular a atividade do exossomo durante o processamento do pré-rRNA.
The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxylterminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Δnop8/GAL::NOP8. Interestingly, Nop8p mediates the association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control exosome function during pre-rRNA processing.

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16

Sikorska, Natalia. "The phosphorolytic activity of the exosome core complex contributes to rRNA maturation in Arabidopsis thaliana." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ070/document.

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L’exosome joue un rôle fondamental dans la dégradation de 3’ en 5’ et la maturation des ARNs chez les eucaryotes. Le "coeur" de l’exosome est composé de 9 sous-unités (EXO9). EXO9 est catalytiquement inactif chez l’homme et la levure, et est associé à deux RNases, Rrp6 et Rrp44, responsables de l’activité exonucléolytique de l’exosome. Mes travaux de thèse démontrent que chez Arabidopsis, le coeur de l’exosome EXO9 possède une activité catalytique intrinsèque. Cette activité est dépendante de la présence de phosphate, produit des nucléosides diphosphates et est réversible. Elle possède de ce fait toutes les caractéristiques d’une activité phosphorolytique. L’activité d’EXO9 est impliquée dans l’élimination de sous-produits de la maturation des ARNr et dans la maturation de l’ARNr 5.8S, deux fonctions typiques de l’exosome. Mes travaux révèlent également que AtRRP44, EXO9 et AtRRP6L2 coopèrent de manière séquentielle pour la maturation de l’ARN 5.8S. Mes travaux de thèse constituent la base de travaux futurs visant à comprendre les rôles de l’activité phosphorolytique de l’exosome chez un organisme eucaryote
The eukaryotic RNA exosome complex is the main 3’-5’ degradation machinery that plays an essential role in RNA decay, quality control and maturation. The exosome core complex (EXO9) is catalytically inert in yeast and humans, and therefore relies on the catalytic activity of associated RNases, Rrp6 and Rrp44. In this study I demonstrated that EXO9 is catalytically active in Arabidopsis. EXO9’s activity is phosphate-dependent, releases nucleoside diphosphates and is reversible, meeting all criteria of a phosphorolytic activity. Importantly, EXO9’s in vivo substrates include the archetypical exosome substrates, rRNA maturation by-products and 5.8S rRNA precursors. My data show that AtRRP44, EXO9 and AtRRP6L2 sequentially cooperate for the processing of 5.8S rRNA. This work sets a basis for studies aiming at further understanding the biological functions of EXO9’s phosphorolytic activity in a eukaryotic organism

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17

Zhao, Lifang. "ECERIFERUM7 subunit of the exosome, SUPERKILLER complex and small RNA species regulate cuticular wax biosynthesis in Arabidopsis thaliana stems." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52668.

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The primary aerial surfaces of higher plants are covered by a continuous hydrophobic lipid layer called the cuticle, which is synthesized by the epidermal cells and provides protection against desiccation and environmental stresses. The cuticle is mainly composed of the cutin polyester matrix and cuticular waxes. Although the biosynthetic pathways of cuticular waxes are relatively well documented, how wax biosynthesis is regulated is not completely understood. The major goal of my thesis was to investigate the ECERIFERUM7 (CER7)-mediated mechanism of regulation of cuticular wax biosynthesis in stems of Arabidopsis thaliana. In particular, I was interested in investigating that how the Arabidopsis CER7 protein, a core component of the exosome complex that determines cellular RNA levels, was involved in this process. CER7 was proposed to degrade an mRNA encoding a repressor of wax biosynthetic gene CER3 to activate CER3 transcription required for stem wax biosynthesis. To identify the CER3 repressor and additional components of CER7 regulatory pathway, I carried out a cer7 suppressor screen and isolated mutants capable of restoring wild-type stem wax loads in the absence of CER7 activity. Characterization of these suppressor mutants and cloning of the affected genes resulted in a series of discoveries. First, cloning of RNA DEPENDENT RNA POLYMERASE 1 and SUPPRESSOR OF GENE SILENCING 3 from the suppressors demonstrated that small interfering RNAs (siRNAs) participate in CER7-mediated regulation of wax formation (Chapter 2). Second, forward genetics and reverse genetics, combined with small RNA sequencing confirmed that trans-acting siRNAs (tasiRNAs) are direct regulators of CER3 gene in CER7-controlled wax biosynthetic pathway (Chapter 3). Third, CER7 and tasiRNA-mediated regulation of CER3 during stem wax deposition requires the SUPERKILLER complex, which is known to be involved in cytoplasmic activities of the exosome in yeast and metazoan (Chapter 4).
Science, Faculty of
Botany, Department of
Graduate

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Mullani, Nowsheen. "An RNA Signature Links Oxidative Stress To Cellular Senescence." Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS560.pdf.

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Le stress oxydatif est l’une des voies menant à la sénescence cellulaire. Bien que les dommages causés par les espèces réactives de l'oxygène aux protéines et à l'ADN soient bien décrits, notre compréhension de la manière dont la transcription peut participer à l'apparition de la sénescence est encore limitée. Au niveau de la transcription, le stress oxydatif entraîne l’accumulation d’ARN promoteurs (ARNAu) et d’ARN amplificateur (ARNs), conséquence de la libération défectueuse du RNAPII de la chromatine, un phénomène connu sous le nom de RNAPII crawling. Nous avons observé que l'exploration de RNAPII était également détectée en aval d'une petite série de gènes connus pour être régulés par HP1Υ au niveau de leur terminaison. L'exploration de ce phénomène a donné un résultat inattendu, en ce sens qu'il a révélé un effet inhibiteur du peroxyde d'hydrogène sur le complexe exosome d'ARN impliqué dans la dégradation des ARN polyadénylés. Le RNAPII rampant a pour résultat la transcription de séquences d’ALU situées au voisinage des promoteurs et amplificateurs et en aval de gènes sans intron et de petites séries de gènes contenant un intron. Comme les séquences ALU contiennent des séquences A codées par le génome, elles doivent normalement être dégradées par l’exosome de l’ARN. Cependant, comme le stress oxydatif inhibe également cette activité d'ARNase, les ARNm contenant des séquences d'ALU transcrites par hasard se stabilisent et sont détectés dans le cytoplasme et même dans les fractions de polysomes. Ce phénomène peut participer à l'apparition de la réponse à l'interféron associée au stress oxydatif
Oxidative Stress is one of the routes leading to cellular senescence. While the damages that reactive oxygen species inflict on proteins and DNA are well described, our insight on how transcription may participate in the onset of senescence is still limited. At a transcriptional level, oxidative stress results in accumulation of promoter RNAs (uaRNAs) and enhancer RNAs (eRNAs) as a consequence of defective release of the RNAPII from the chromatin a phenomenon known as RNAPII crawling. We observed that RNAPII crawling was also detected downstream of a small series of genes known to be regulated by HP1Υ at the level of their termination. Exploring this phenomenon yielded an unexpected result in the sense that it revealed an inhibiting effect of hydrogen peroxide on the RNA exosome complex involved in degradation of polyadenylated RNAs. The crawling RNAPII results in the transcription of ALU sequences located in the neighborhood of promoters and enhancers and downstream of intron-less genes and of small series of intron-containing genes. As ALU sequences contain genome encoded A tracts, they should normally be degraded by the RNA exosome. Yet, as oxidative stress also inhibits this RNAse activity, mRNAs containing serendipitously transcribed ALU sequences get stabilized and are detected in the cytoplasm and even polysome fractions. This phenomenon may participate in the onset of the interferon response associated with oxidative stress

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Zubiate, Fernando Alexis Gonzales. "Estudo de interações entre subunidades do exossomo e com outras proteínas celulares em Saccharomyces cerevisiae." Universidade de São Paulo, 2001. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-23012019-094516/.

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Em Saccharomyces cerevisiae, Rrp43p é uma proteína que faz parte do exossomo, um complexo multiproteíco que atua no processamento de snoRNAs, snRNAs e rRNAs e na degradação de mRNA. O exossomo está envolvido nesses processos através de uma ação 3\'→5\' exonucleolítica. Este complexo é composto por onze subunidades em S. cerevisiae e cada uma dessas subunidades tem uma ação predominante nos diferentes processos em que o complexo participa. Por estar envolvido diretamente na maturação dos rRNAs e alguns snoRNAs e snRNAs, assim como na degradação de mRNAs, o exossomo tem um papel importante no controle de expressão gênica. Com o objetivo de entender melhor a função da subunidade do exossomo Rrp43p, e conseqüentemente do complexo, nas modificações do RNA, realizamos estudos de interação entre proteínas através do método de \"two hybrid\", que permite analisar interações in vivo entre duas proteínas, convertendo-se assim, em uma ferramenta importante nestes estudos. Utilizando Rrp43p como \" isca\" estudamos interações com outras proteínas expressas em Saccharomyces cerevisiae, na procura de proteínas envolvidas em alguns eventos de processamento de RNA, que pudessem ajudar a esclarecer em maior detalhe o papel do exossomo na célula. Também examinamos interações da Rrp43p com os demais componentes do exossomo para determinar a possível estrutura deste complexo. Os resultados obtidos demonstram que Rrp43p interage com somente uma outra subunidade do exossomo, Rrp46p. A força desta interação, quando quantificada através do nível de expressão de um gene repórter, é compatível com o fato dessas proteínas formarem parte de um complexo. Estes dados constituem resultados inéditos a respeito da interação entre subunidades do exossomo. Os resultados evidenciam também a interação entre Rrp43p e uma proteína com função ainda não caracterizada em levedura (aqui denominada 137p). Esta interação foi detectada através do sistema do duplo híbrido, e depois confirmada por co-imunoprecipitação. A determinação da função desta nova proteína poderá ampliar as ferramentas de estudo da função e controle de atividade de Rrp43p e do exossomo.
In the yeast Saccharomyces cerevisiae, Rrp43p is one of the eleven subunits of the exosome, a complex involved in the processing of snoRNAs, snRNAs and rRNAs, and in mRNA degradation. The exosome participates in these processes through a 3\'-to-5\' exonucleolytic activity. Each of the eleven subunits is predominately active in one or few of the processes in which the complex takes part. Since the exosome is involved directly in rRNAs, and in some snRNAs and snoRNAs maturation, as well as in mRNA degradation, it plays an important role on the control of gene expression. Aiming to a better understanding of the Rrp43p subunit function on RNA processing, we started a screening for Rrp43p-interacting proteins through the yeast two hybrid system. In this study we expected to find proteins interacting with Rrp43p, which were involved in some aspects of RNA processing, and would improve the current knowledge on the exosome function. In order to obtain more information about the complex structure, we have also studied the interactions between Rrp43p and the other exosome subunits. The results shown here demonstrate that Rrp43p interacts with only one other exosome subunit, Rrp46p. These results can help elucidate the final exosome structure. We also found the interaction of Rrp43p with a protein of yet uncharacterized function (here named 137p). This interaction, identified in the two hybrid system, was also confirmed through co-immunoprecipitation analysis, and the study of 137p function might bring new insights on Rrp43p function and control.

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20

川本, 崇仁. "U snRNA の成熟と分解の分子機構の研究." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263510.

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21

Hocquet, Clémence. "Etude du rôle de Condensine dans le contrôle de l'expression génique chez la levure Schizosaccharomyces pombe." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN037.

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Condensine est un complexe protéique organisateur du génome qui conduit l’assemblage des chromosomes et promeut leur transmission fidèle en anaphase. De nombreuses études ont rapporté des changements dans les niveaux des ARNs cellulaires quand Condensine est défaillante, suggérant un rôle pour Condensine dans la régulation de l’expression génique. Cependant, les mécanismes sous-jacents sont demeurés énigmatiques, et l’on ignore dans quelle mesure le rôle joué par Condensine dans l’expression génique est lié ou non à sa fonction dans l’organisation des chromosomes. Lors de ma thèse, j’ai étudié l’activité de Condensine dans la régulation de l’expression génique en utilisant la levure S. pombe comme organisme modèle. Contrairement à l’idée communément admise, mes résultats montrent que Condensine ne joue aucun rôle direct dans le maintien du transcriptome, ni en interphase, ni en mitose chez cette levure. En accord avec les études précédentes, j’observe des changements de niveau et de qualité des ARNs dans les cellules mutantes pour Condensine au sortir de la mitose ; des ARNs non codants et des ARNs aberrants, étendus en 3’, s’accumulent. En revanche, je démontre que ces changements sont la conséquence de défauts de transmission des chromosomes en anaphase. L’inactivation de Condensine cause la non-disjonction de l’ADN ribosomique et du nucléole, entrainant une déplétion de l’ARN-exosome des cellules filles, lesquelles accumulent alors des ARNs normalement dégradés par l’ARN-exosome. De façon cruciale, je montre qu’empêcher les anomalies de migration des chromosomes restaure une expression normale des gènes malgré l’inactivation de Condensine, démontrant que c’est l’instabilité chromosomique qui est source des changements d’expression génique observés quand Condensine est défaillante, et non le complexe Condensine en tant que tel. Ce travail remet en question le concept de régulation de l’expression génique par les complexes Condensine et appelle à la prudence lorsque l’on cherche à étudier les fonctions de ces complexes en dehors de la condensation de la chromatine en mitose
Condensin is a genome organiser that shape chromosomes and promote their accurate transmission in anaphase. Several studies have related changes in RNA level when Condensin is defective, suggesting that the complex has also a role in gene expression. However, the mechanisms have remained enigmatic and we still don’t know to what extent it is related to its role in chromosome organization. During my thesis, I studied the role played by Condensin in the regulation of gene expression using S. pombe as a model system. In contrast to previous studies, my results provide compelling evidence that Condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis in this yeast. Accordingly to previous studies, I observed changes in RNA level in cells mutated for Condensin; non coding and 3’ extended RNA accumulate. However, I showed that the changes in gene expression in post-mitotic fission yeast cells that result from Condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite Condensin inactivation. Thus, chromosome instability, rather than a direct role of Condensin in the transcription process, changes gene expression. This work challenges the concept of gene regulation by canonical Condensin complexes and ask for caution when studying Condensin role outside chromosome condensation in mitosis

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Cepeda, Leidy Paola Paez. "Caracterização da função molecular de Nop53 e de seu papel no controle do exossomo em Saccharomyces cerevisiae." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22112017-140024/.

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Nop53 e uma protena nucleolar, conservada evolutivamente e essencial na levedura Saccharomyces cerevisiae para a biogênese da subunidade maior do ribossomo, 60S. O principal fenotipo causado pela repressão da expressão de Nop53 e o acumulo do intermedi ario de processamento de pre-Rrna, 7S, que tambem e substrato do complexo exossomo na formação do rRNA maduro 5:8S. Nop53 interage diretamente com a subunidade do exossomo Rrp6 e com a subunidade Mtr4 do co-ativador do exossomo TRAMP. O objetivo principal deste trabalho foi o de analisar como a interação entre Nop53 e o exossomo pode modular a atividade deste ultimo. Para isso, foram utilizados metodos bioqumicos, geneticos e de biologia molecular. Os resultados mostrados aqui demonstram que a depleção de Nop53 faz com que mais protenas ribossomais, principalmente da subunidade maior, sejam co-imunoprecipitadas com o core do exossomo, sugerindo que Nop53 possa ter um papel na liberação do exossomo da subunidade pre-60S depois da formação do rRNA maduro 5:8S. Esta hipotese foi conrmada atraves da separação de complexos por centrifugação em gradiente de glicerol, que mostrou a presenca de subunidades do exossomo em complexos maiores na ausência de Nop53, provavelmente correspondendo a partculas pre-ribossomais. Co-imunoprecipitação de RNA com o exossomo na ausência de Nop53 tambem conrmou uma maior associação deste complexo com o pre-rRNA 7S. Como tambem mostrado aqui, alem de interagir com Rrp6, Nop53 interage com subunidades do core do exossomo e a superexpressão de uma destas subunidades, Rrp43, complementa parcialmente a ausência de Nop53 na celula. Estes resultados levaram a conclusão de que Nop53 pode recrutar o exossomo para a partcula ribossomal pre-60S para a maturação do pre-rRNA 7S a 5:8S, e atue tambem na liberação do exossomo, possivelmente atraves de sua interação com a helicase Mtr4.
Abstract Nop53 is a nucleolar, conserved and essential protein in the yeast Saccharomyces cerevisiae, involved in the biogenesis of the large ribosomal subunit 60S. The main phenotype of the depletion of Nop53 in yeast cells is the accumulation of the prerRNA processing intermediate 7S, which is also the substrate of the exosome complex for the formation of the mature rRNA 5:8S. Nop53 directly interacts with the exosome subunit Rrp6, and with the subunit Mtr4 of the TRAMP complex, an exosome co-activator. The main objective of this work was the analysis of the interaction between Nop53 and the exosome and the identication of the mechanism through which Nop53 regulates the exosome activity. The results shown here demonstrate that the depletion of Nop53 leads to a more stable association of the exosome with the pre-60S ribosome particle, as determined by co-immunoprecipitation of proteins with one of the exosome core subunits, and by fractionation of complexes through glycerol gradients. These results suggested that Nop53 could play a role in the release of the exosome after the formation of the mature rRNA 5:8S. This hypothesis was conrmed through the co-immunoprecipitation of pre-rRNA 7S with the exosome in the absence of Nop53. In addition to the interaction with the exosome subunit Rrp6, as shown here, Nop53 also interacts with core subunits of the complex. Interestingly, overexpression of one of these subunits, Rrp43, partially complements the depletion of Nop53. These results led to the conclusion that Nop53 may recruit the exosome to the pre-60S particle for the maturation of the pre-rRNA 7S to the mature 5:8S, but Nop53 may also be involved in the release of the exosome, possibly through its interaction with the helicase Mtr4.

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Dangin, Mathieu. "Contrôle de la différenciation sexuelle de la levure Schizosaccharomyces pombe par un ARN non-codant et la protéine de liaison à l’ARN Mmi1." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV079/document.

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Au cours des cinq dernières années l’existence d’un contrôle de la transcription par les ARN non-codants longs (lncRNAs) a été décrite dans une large variété d’eucaryotes. Cependant, les mécanismes par lesquels les lncRNAs régulent la transcription restent en grande partie méconnus. Les premiers travaux effectués dans le cadre de cette thèse ont participé à la caractérisation du mécanisme mis en jeu par un lncRNA, nommé nam1, dans le contrôle de l’entrée en différenciation sexuelle chez la levure Schizosaccharomyces pombe. Il a ainsi été montré qu’au cours de sa synthèse le lncRNA nam1 est ciblé par la protéine de liaison à l’ARN Mmi1 et une machinerie de surveillance des ARN qui comprend l’exosome, un complexe de dégradation des ARN conservé au cours de l’évolution. La fixation de Mmi1 au lncRNA nam1 contrôle la terminaison de la transcription de nam1 et empêche ainsi la transcription de se poursuivre et d’interférer alors avec la transcription du gène situé en aval (codant pour une MAP kinase essentielle à l’entrée en différenciation). Les travaux suivant montrent l’implication dans ce mécanisme de la protéine Cti1, un des co-facteurs connus de l’exosome. Fait marquant, ces travaux rapportent aussi l’existence d’un mode de production inédit pour un lncRNA. En effet, ils révèlent que la transcription non-interrompue d’un gène codant conduirait à la production d’un ARN bi-cistronique. La maturation co-transcriptionnelle de cet ARN bi-cistronique produirait, d’un côté, un ARN messager et, de l’autre, le lncRNA nam1. Enfin, ils ont permit la caractérisation initiale d’un nouveau composant de la machinerie de surveillance des ARN recrutée sur nam1 par Mmi1. Ainsi, dans leur ensemble, ces travaux contribuent à une meilleure connaissance des mécanismes pouvant être mis en jeu par un lncRNA et agissant en cis pour réguler l’expression génique et, à travers elle, des processus cellulaires majeurs, tel que la différenciation cellulaire. De plus, ils décrivent un nouveau mécanisme de biogénèse d’un lncRNA
Over the last five years, the control of transcription mediated by long non-coding RNAs (lncRNAs) has been reported to take place in a wide variety of eukaryotes. However, the mechanisms by which lncRNAs regulate transcription remain relatively poorly described. The first work conducted in the context of this PhD thesis has contributed to the characterization of the mechanism used by a lncRNA, named nam1, to control entry into sexual differentiation of the fission yeast Schizosaccharomyces pombe. It was shown that, while the lncRNA nam1 is being produced, it is targeted by the RNA binding protein Mmi1 and a RNA surveillance machinery that includes the exosome, a conserved complex throughout evolution. The binding of Mmi1 to nam1 lncRNA controls the termination of transcription of nam1, which prevents this non-coding transcription from interfering with the transcription of the downstream gene, coding for a MAP kinase essential to entry into differentiation. The following work shows the importance of the protein Cti1, one of the known co-factor of the exosome, in the nam1-dependent control of sexual differentiation. Remarkably, it also strongly suggests the existence of a new way of producing a lncRNA. Indeed, it reveals that read-through transcription of a protein-coding gene leads to the production of a bi-cistronic RNA, which is co-transcriptionally matured to produce on one side a messenger RNA and on the other side the lncRNA nam1. Finally, this work initiated the characterization of a new component of the RNA surveillance machinery targeting nam1. Collectively, this work brings several insights into the mechanisms used by cis-acting lncRNAs to regulate gene expression and, thereby, major cellular processes such as cell differentiation. Moreover, it also provides insights into the biogenesis of lncRNAs by reporting a new mode of production of lncRNAs

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Mamolen, Megan Christine. "DROSOPHILA MELANOGASTER DIS3 IS A DYNAMIC ENDO- AND 3’ to 5’ EXORIBONUCLEASE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1278525341.

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Schillewaert, Stéphanie. "Etude de la maturation et de l'assemblage du ribosome eucaryote: caractérisation fonctionnelle de nouveaux facteurs trans-." Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209826.

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La synthèse du ribosome est un processus compliqué, très hiérarchisé et essentiel à toutes les cellules vivantes. La complexité de ce processus tient notamment au fait que les différentes étapes de la biogenèse du ribosome eucaryote sont temporellement et spatialement organisées dans des compartiments cellulaires différents (le nucléole, le nucléoplasme et le cytoplasme). Il est toutefois connu que le pré-ARNr 35S (le précurseur de trois des quatre ARNr, les ARNr 18S, 5.8S et 25S) est pris en charge dès sa synthèse par des facteurs impliqués dans sa maturation. Ainsi, la formation d’un ribosome requiert l’association, sur le transcrit naissant, des facteurs de synthèse, au nombre de 400. Ces facteurs essentiels interagissent transitoirement avec l’ARNr et ne font pas partie des particules ribosomiques matures impliquées dans la traduction. Leur rôle est d’assister le remodelage constant du pré-ribosome et le processus d’assemblage des sous-unités.

Parmi ces facteurs de synthèse, nous avons caractérisé en détail, chez la levure et chez l’homme, la protéine Las1 impliquée dans la maturation des deux extrémités de l’ITS2, séquence qui sépare les ARNr 5.8S et 25S/28S. Chez la levure, en absence de la protéine Las1, les analyses de profils de polysomes révèlent un déficit de sous-unité 60S et l’apparition d’« halfmères ». Les techniques de purification d’affinité et de gradient de sédimentation nous indiquent que Las1 est associée aux pré-ribosomes 60S et qu’elle interagit avec de nombreux facteurs de synthèse de la petite, de la grande sous-unité ou des deux. De plus, Las1 copurifie avec des pré-ribosomes qui contiennent aussi les exoribonucléases 5’-3’ Rat1/Rai1 et Xrn1. Rai1 coordonne la maturation aux deux extrémités de l’ARNr 5.8S. Nous suggérons que Las1 appartient à un macrocomplexe connectant spatialement des sites de clivages éloignés sur la séquence primaire du pré-ARNr qui seraient rapprochés suite au reploiement de l’ITS2.

Un autre aspect de ce travail de thèse consiste en l’étude de l’assemblage des particules ribonucléoprotéiques et plus spécifiquement du pré-ribosome et des sous-unités ribosomiques eucaryotes. Nous avons utilisé la technique d’immunoprécipitation de chromatine (Ch-IP) pour caractériser l’assemblage d’une structure appelée le « SSU processome ». Celui-ci correspond à un pré-ribosome en formation ainsi que l’assemblage des protéines ribosomiques sur l’ARNr naissant.

Enfin, nous avons étudié le rôle d’une plateforme d’activation de méthyltransférases d’ARN et de protéines, la protéine Trm112 dans la ribogenèse. Nous avons montré que chez la levure, Trm112 est impliquée dans la synthèse du ribosome et dans la progression de la mitose. En absence de cette protéine, les pré-ARNr sont dégradés par un mécanisme de surveillance. Trm112 copurifie avec plusieurs facteurs de synthèse du ribosome dont la méthyltransférase Bud23, impliquée dans la modification post-transcriptionnelle de l’ARNr18S. Trm112 est requise pour cette méthylation et nous postulons que la protéine Bud23 est incapable de se lier aux pré-ribosomes en l’absence de Trm112.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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26

Menino, Glaucia Freitas. "Estudo do exossomo de Archaea e de sua interação com a proteína reguladora PaNip7." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-06042016-143157/.

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O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo.
The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.

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Balaratnam, Sumirtha. "BIOGENESIS AND FUNCTIONAL APPLICATIONS OF PIWI INTERACTING RNAs (piRNAs)." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1531753741509242.

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28

Zhang, Elodie. "Etude de facteurs impliqués dans le contrôle-qualité de l'expression des gènes, chez Saccharomyces cerevisiae." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066500/document.

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La régulation et le contrôle-qualité de l'expression génique permettent respectivement de maintenir un équilibre entre synthèse et dégradation des ARNm répondant aux besoins cellulaires et d'empêcher l'expression d'ARNm ou protéines aberrants potentiellement toxiques. Pour mieux comprendre ces processus cytoplasmiques, je me suis intéressée à Jlp2, Tac4 et Ska1, trois protéines ayant des liens physiques ou fonctionnels avec des acteurs du contrôle-qualité des ARNm et peptides appartenant aux complexes RQC et SKI. Jlp2 montre des liens de létalité synthétique avec les complexes RQC et SKI mais son absence n'altère pas le " NonStop mRNA Decay ". Elle pourrait donc être impliquée dans une autre voie de contrôle dépendante des complexes RQC et SKI. Tac4 est une ARN hélicase putative associée aux ribosomes, au niveau de l'hélice H16 de l'ARNr 18S comme son homologue putatif mammifère DHX29. Elle interagit également au niveau de régions 3'UTR d'ARNm. Ces observations suggèrent que Tac4 pourrait être impliquée dans la réinitiation de la traduction et le sauvetage de ribosomes non-dissociés récemment identifiés dans la région 3'UTR d'ARNm. Enfin, nous avons identifié Ska1, une protéine appartenant à une nouvelle sous-population de complexes SKI. Nos données suggèrent que ce complexe SKI-Ska1 est impliqué dans la dégradation de transcrits dépourvus de ribosome. Nous proposons un modèle selon lequel ce complexe SKI-Ska1 agirait durant la dégradation de 3'UTR avec l'exosome, puis en arrivant dans la région codante et en rencontrant un ribosome, Ska1 se dissocierait du complexe pour lui permettre d'interagir directement avec le ribosome et poursuivre la dégradation 3'-5' de l'ARN
Mechanisms responsible for the regulation of gene expression and its quality-control are required, respectively for maintaining an equilibrium between mRNA synthesis and degradation and to prevent synthesis of aberrant mRNAs and proteins potentially toxic for the cells. To better understand these quality-control processes, I studied three factors, Jlp2, Tac4 and Ska1, with physical or functional links described with factors involved in mRNA and protein quality-control, the RQC and SKI complexes. Jlp2 shows synthetic lethality with the RQC and SKI complexes but its deletion has no effect on the NonStop mRNA Decay, suggesting that Jlp2 could be implicated in another control pathway linked to the RQC and SKI complexes. Tac4 is a putative RNA helicase bound to ribosomes, on the 18S rRNA H16 helix, as its mammalian putative homolog DHX29. DHX29 plays a role in translation initiation but surprisingly, Tac4 interacts, in addition to ribosomes, with mRNA 3’UTRs. These observations suggest that Tac4 could be implicated in translation reinitiation and rescue of non-dissociated-ribosomes, recently described within mRNA 3’UTRs. Finally, we identified Ska1, a new factor associated to a SKI complex subpopulation. Our observations suggest that the SKI-Ska1 complex is implicated in the degradation of transcripts devoid of ribosomes. It suggests a model by which the SKI complex would proceed in two steps. First, the SKI-Ska1 complex could assist the exosome to degrade 3’UTR regions of RNAs and then, when its reaches the coding region and encounter a ribosome, Ska1 would leave the complex and allow it to interact directly with ribosomes to proceed further in the 3’-5’ RNA degradation

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Ruidant, Sabine. "Visualisation des Arbres de Noël de Miller par immunoprécipitation de chromatine (ChIP) et mise en évidence d'un mécanisme de surveillance nucléolaire des ARN ribosomiques." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210384.

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Les terminal balls qui constituent des complexes de maturation sont détectées

à l’extrémité 5’-terminale des transcrits ribosomiques naissants dans tous les organismes

eucaryotes inspectés à ce jour ;générant les images de référence en « arbres de Noël ».

La compaction séquentielle des « terminal balls », à présent également dénommées

« SSU-processome », reflète les étapes d’assemblage co-transcriptionnel des ribosomes.

Au cours de ma thèse, j’ai développé une stratégie expérimentale basée sur

l’immunoprécipitation de chromatine (ChIP) qui m’a permis de valider, et ce pour la

première fois, in vivo la structure des branches des arbres de Noël (en particulier un

rapprochement du « SSU-processome » à l’extrémité 5’- du gène encodant l’ARNr 25S).

Notre stratégie nous permet également d’aborder la composition moléculaire des « arbres

de Noël ».

La biogenèse du ribosome est un processus complexe et dynamique dont la

finalité est la synthèse et l’assemblage de 4 molécules d’ARN et de ~80 protéines

ribosomiques dans un processus qui requiert l’intervention transitoire et concertée de non

moins de 400 facteurs de maturation. D’une telle complexité a récemment émergé le

concept de l’existence de modules pré-assemblés autonomes de facteurs de maturation.

Dans le cas du « SSU-processome », les trois sous-complexes UTP-A, UTP-B, UTP-C

ont d’ores et déjà été décrits. L’existence de tels sous-complexes renforce la notion d’un

mécanisme d’assemblage hautement hiérarchisé. En effet, il s’est avéré que l’extrémité

5’- du transcrit naissant est initialement liée par le sous-complexe UTP-A dans une étape

qui est un pré-requis indispensable au recrutement et à l’assemblage des autres

composants du « SSU-processome ». Avec autant d’étapes distinctes dans le processus

d’assemblage, la possibilité d’erreur est conséquente, d’où l’importance de l’existence de

mécanismes de contrôles de qualité.

Toutes les protéines constituant le « SSU-processome » sont requises au clivage

des précurseurs d’ARN ribosomique. Préalablement à mon travail, les 7 sous-unités

protéiques du complexe UTP-A avaient, en outre, spécifiquement été impliquées dans la

synthèse de l’ARN, c’est-à-dire dans la fonction de l’ARN polymérase I. Ceci leur a

conféré leur seconde appellation de tUTP, pour transcription UTP, et offert les prémices

de l’existence d’une interface physique et fonctionnelle entre les machineries de synthèse

et de maturation des ARNr. Au cours de ma thèse, j’ai démontré qu’il n’en est rien. Une

inspection minutieuse m’a en effet révélé que les tUTP/UTP-A ne sont nullement

requises à la synthèse des ARN ribosomiques mais bien à leur stabilité. Cette observation

m’a mené à proposer que la cellule a développé au cours de l’évolution un mécanisme de

contrôle de qualité par lequel elle s’assure de l’intégrité des étapes initiales d’assemblage

(liaison du complexe UTP-A ). Mon postulat est qu’en l’absence de la liaison de ces

facteurs de maturation précoces, les ARN sont rapidement dégradés par un mécanisme

que nous avons dénommé « Death by Default (DBD) » par l’activité de surveillance

nucléolaire exercée par le complexe de polyadényaltion TRAMP et d’exoribonucléases

3’-5’ l’ Exosome.
Doctorat en sciences, Spécialisation biologie moléculaire
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30

Li, Zhefeng. "Ligand Displaying Exosomes by Arrowtail RNA Nanoparticles for siRNA Delivery and Scale-up Production." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1590062022556383.

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31

Yazbek, Hanna Marcelino. "Exosomal RNA as a source of urine biomarkers for prostate cancer." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/66563/.

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Introduction: In this study we exploited the recent development of methods that have enabled the analysis of RNA present in urine exosomes of prostate cancer patients. We report RNA expression patterns that contain diagnostic and prognostic information for prostate cancer, and association with response to hormone treatment. Methods: First catch urine following digital rectal examination were collected from 662 men. 3 groups of patients were used: Low, Intermediate, and High-risk according to NICE stratification criteria, and two control groups: benign and advanced disease. 50-gene transcript expression analysis using NanoString technology was performed on 192 samples. Exosomal RNA Next-Generation Sequencing was performed on 18 samples for novel biomarker discovery. Results: Expression analysis showed that PCa-specific transcripts such as TMPRSS2/ERG fusion transcripts were identifiable in exosomes from PCa urine samples. LPD analysis highlighted expression levels of 15 transcripts with diagnostic potential (significantly up-regulated in cancer samples in comparison to benign control) and 17 transcripts with prognostic potential (differentialy expressed in high risk and advanced disease in comparison to lower grade disease). I also report two gene transcripts (SERPINB5/Maspin, HPRT) that were significantly differentially expressed in patients who failed to respond to hormone deprivation therapy for high risk/metastatic disease. Three genes (STEAP4, ARexons4_8 and NAALADL2) were significantly differentially expressed in patients who relapsed within 12 months of hormone treatment initiation. Next-Generation Sequencing of twenty samples identified 45 genes to be significantly differentially expressed between non-cancer and cancer samples (28 were up regulated and 17 down regulated). 33 out of the 45 genes showed a significant linear trend in association with cancer risk. Conclusions: Urine Exosomal RNA contains PCa specific transcripts. Gene expression analysis and Next Generation Sequencing identified genes that are significantly differentially expressed between cancer and non-cancer cases as well as prognostic genes and genes that can predict response to hormone treatment.

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Giunta, Michele. "Exosomal protein deficiencies : how abnormal RNA metabolism results in childhood-onset neurological diseases." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3669.

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RNA metabolism is of critical importance for normal cellular functions and needs to be finely tuned in order to maintain stable conditions within the cell. The exosome complex is the most important RNA processing machinery, responsible for the correct processing of many different types of RNAs and interacting with different co-factors which bind and carry specific subtypes of RNA for degradation to the complex. Mutations in exosome complex subunits (EXOSC3, EXOSC8) were reported to cause severe childhood onset complex neurological disorders presenting with pontocerebellar hypoplasia type 1 (PCH1), spinal muscular atrophy (SMA) and central nervous system hypomyelination. We have recently identified a homozygous pathogenic mutation in RNA Binding Motif Protein 7 RBM7, a subunit of the nuclear exosome targeting (NEXT) complex in a single patient with SMA-like phenotype and proved that RBM7 is a novel human disease gene related to the exosome complex. In order to understand the disease mechanism in RBM7 deficiency and to explore the role of exosome complex in neurodevelopment, we performed gene expression studies (RT-PCR, RNA sequencing) in human cells of patients carrying mutations in EXOSC8 and RBM7. Furthermore we performed functional studies in zebrafish (D. rerio) by morpholino oligonucleotide mediated knock-down of rbm7, exosc8 and exosc3 and also by introducing pathogenic mutations in exosomal protein genes in zebrafish embryos by the CRISPR/Cas9 system. We showed that mutations in RBM7 and EXOSC8 mutant fibroblasts cause differential expression of several different transcripts, 62 of them being shared between the two cell lines. Altered gene expression of some AU-rich element containing genes may potentially contribute to the clinical presentation. Knock-down of rbm7, exosc8 and exosc3 caused impaired neurodevelopment in zebrafish, illustrated by abnormal growth of motor neuron axons and failure to differentiate cerebellar Purkinje cells. RT-PCR analysis in zebrafish showed a dramatic increase in expression of atxn1b (an AU-rich element containing homolog of the human ATXN1 gene) in rbm7, exosc8 and exosc3 downregulated fish, which may be responsible for the cerebellar defects. We have successfully introduced several germline mutations with CRISPR/Cas9 technology in rbm7. Phenotype of the F1 mutants is milder than what observed with the morpholino oligonucleotide injected fish. Mutants at a closer look do not show any morphological defect but further experiment may indicate similar characteristics to the morphants, although more iv subtle. Further studies on the CRISPR/Cas9 generated zebrafish models will extend our knowledge on the disease mechanisms caused by defective RNA metabolism.

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Chitiprolu, Maneka. "Novel Regulatory Mechanisms of Autophagy in Human Disease: Implications for the Development of Therapeutic Strategies." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38441.

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The dysfunction of autophagy pathways has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these pathological autophagy mechanisms is essential to gain insights into the underlying disease mechanisms, identify novel biomarkers, and develop targeted therapies. In this thesis, I present three manuscripts that investigate the regulatory mechanisms of autophagy machinery in human diseases. In the first manuscript (Chitiprolu et al., 2018), we investigated the mechanism of p62-mediated selective autophagic clearance of RNA stress granules implicated in Amyotrophic Lateral Sclerosis (ALS). Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. By employing mass spectrometry, high resolution imaging and biochemical assays, we demonstrated that the autophagy receptor p62 associates with C9ORF72 to eliminate stress granules by autophagy. This requires p62 to associate with proteins that are symmetrically methylated on arginines. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients. The second manuscript (Guo, Chitiprolu et al., 2014) describes the mechanism by which autophagy degrades retrotransposon RNA from both long and short interspersed elements, thereby preventing new retrotransposon insertions into the genome. By employing quantitative imaging tools, we demonstrated that retrotransposon RNA localizes to RNA granules that are selectively degraded by the autophagy receptors NDP52 and p62. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. This suggests a mechanism for the increased tumorigenesis upon autophagy inhibition and therefore a role for autophagy in tempering evolutionary change. Finally, the third manuscript (Guo, Chitiprolu et al., 2017) examines the intersection of autophagy machinery with exosome release and function in cancer metastasis. By employing dynamic light scattering, Nanosight particle tracking, electron microscopy, super-resolution imaging and Western blotting, we robustly quantified exosome identity and purity in multiple cell lines. We demonstrated that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings delineate autophagy-independent pathways by which autophagy-related genes can contribute to metastasis. Taken together, data presented in the three manuscripts highlight the molecular mechanisms of autophagy core machinery proteins and selective receptors such as Atg5, p62 and NDP52, in the pathogenesis of cancer and neurodegeneration. In these diseases characterized by mutations in autophagy pathways, the mechanisms we uncover provide insights into their causes and serve as potential therapeutic targets.

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Ipas, Hélène. "Contingent microARN des exosomes, diagnostic et physiopathologie des gliomes." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00986111.

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Les tumeurs gliales du cerveau et en particulier les glioblastomes sont des tumeurs de très mauvais pronostic. Les paramètres qui contrôlent des phénotypes comme l'agressivité, la migration, ou la chimio-résistance de ces tumeurs sont mal connus. Dans ce contexte tumoral, il est envisagé que les microARN (ARN non-codants d'une vingtaine de bases) soient des acteurs essentiels des phénomènes de modification phénotypique parce qu'ils sont capables d'orchestrer l'expression de nombreux gènes. Nous avons montré que les microARN sont des marqueurs tissulaires précieux pour le diagnostic permettant de différencier les deux types principaux de gliomes à partir de prélèvements tumoraux. Nous avons aussi observé que plusieurs microARN sont, en outre, sécrétés par les cellules gliales saines ou cancéreuses au sein de microvésicules appelées exosomes. Le contenu en ARN de ces exosomes a été caractérisé par analyse moléculaire transcriptomique (ARN messagers et microARN) par techniques d'hybridation sur puces à ADN Affymetrix. Les profils ARN exosomaux sains et cancéreux sont distincts, mais ils ne reflètent pas intégralement le profil ARN des cellules dont ils sont issus. Des conditions de stress hypoxique ou l'utilisation de composés pharmacologiques (GW4869 et 5-aza-2'-désoxycitidine) n'affectent pas la quantité d'exosomes produite par la lignée de glioblastome (U87) en culture. Les profils ARN sont cependant modifiés, et le contenu des exosomes produits semble donc être un mécanisme actif et régulé. Enfin, des exosomes cancéreux incubés avec des cellules saines ont très peu d'effet sur le phénotype de celles-ci. Les microARN tissulaires et exosomaux seraient donc des acteurs importants de la physiopathologie du gliome et de sa progression, dont les rôles restent encore à préciser.

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Tavares, José Roberto. "Estudo das interações entre as subunidades do complexo exossomo em Saccharomyces cerevisiae." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07102016-144349/.

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O exossomo é um complexo de exorribonucleases composto por onze proteínas envolvidas no processamento de rRNA, snRNAs, snoRNAs e na degradação de mRNAs. Todas as subunidades do complexo possuem possíveis domínios de RNase porém apenas quatro delas já tiveram a sua atividade de exorribonuclease caracterizada in vitro. Este complexo foi identificado inicialmente em levedura, sendo também encontrado em outros organismos eucarióticos e também em archaea. O estudo das interações entre suas subunidades resultou em modelos estruturais para o exossomo de H sapiens e T. brucei. A despeito do exossomo ter sido caracterizado em levedura, um estudo das possíveis interações entre as subunidades neste organismo ainda não foi realizado. Com a proposta de identificar estas interações em levedura, neste trabalho foi usado o sistema do duplo híbrido, uma poderosa ferramenta para analisar as interações proteína-proteína. Os resultados obtidos através deste sistema mostraram que a subunidade Rrp4p interagiu com as proteínas Rrp41p, Rrp44p, Mtr3p e Rrp6p. A subunidade Rrp41p mostrou uma forte interação com Rrp45p e a subunidade Rrp42p com Mtr3p. A proteína Rrp45p interagiu com Rrp41p e Rrp43p. A proteína Mtr3p interagiu com Rrp4p, Rrp42p e com Csl4p. Em nossos testes a subunidade Rrp40p não mostrou qualquer interação com as outras subunidades do complexo. Outras proteínas envolvidas no processamento RNA como Noplp, Nop58p, Nop8p e Lsm8p também foram testadas para interações com as subunidades do exossomo e mostraram que interagem com Rrp4p e Mtr3p. Estes resultados inéditos trouxeram novas informações para o futuro modelo estrutural do exossomo e novos da dos sobre a participação deste complexo no processamento de RNA. A determinação da sua estrutura pode contribuir para entender a versatilidade deste complexo em vários processos nos quais ele está envolvido na célula.
Exossome is a complex of exorribonucleases composed for eleven proteins involved in the processing of rRNA, snRNAs, snoRNAs and in the degradation of mRNAs. All the subunits of this complex possess possible RNase domains, however only four of them already had its exorribonucleases activity of characterized in vitro. This complex was identified initially in yeast, being also found in other eukaryotes organisms and also in archaea. The study of the interactions between its subunits resulted in structural models for the exossome of H. sapiens and T. bracei. The spite of exossome has been previously characterized in yeast a study of the possible interactions between the subunits in this organism was still not carried through. With the proposal of identifying these interactions in yeast, we used this work system of yeast two hybrid, a powerful tool analyze protein-protein interactions. The results obtained through this system showed that the subunit Rrp4p internet with the proteins Rrp41p, Rrp44p, Mtr3p and Rrp6p. The subunit Rrp41p showed to one strong interaction with Rrp45p and the subunit Rrp42p with Mtr3p. The Rrp45p protein interacted with Rrp41p and Rrp43p. The Mtr3p protein interacted with Rrp4p, Rrp42p and with Cs14p. In our tests the subunit Rrp40p did not show any interaction with the other subunits of the complex. Other proteins involved in processing RNA as Nop1p, Nop58p, Nop8p and Lsm8p had also been tested for interactions with the subunits of the exossome and had shown that they internet with Rrp4p and Mtr3p. These results had brought new inforrnation for a future structural model of the exossome and new data on the participation of this complex in the processing of RNA. The deterrnination of its structure can contribute to better understand the versatility of this complex in the various processes in which it is involved within the cell.

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Moreira, Carine Lais. "Identificação do complexo exossoma de RNA de Trypanosoma rangeli e caracterização da subunidade associada EAP3." reponame:Repositório Institucional da UFSC, 2017. https://repositorio.ufsc.br/xmlui/handle/123456789/176796.

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Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2017.
Made available in DSpace on 2017-06-27T04:22:24Z (GMT). No. of bitstreams: 1 346415.pdf: 1490832 bytes, checksum: 7cfa2ebb03527d549295fed4e02c78de (MD5) Previous issue date: 2017
Exossoma de RNA é um complexo multiproteico composto por 12 subunidades, que possui atividade catalítica no sentido 3 ? 5 e está envolvido no processamento e degradação de vários tipos de RNAs. As subunidades RRP6 (Ribossomal RNA Processing 6) e RRP44 (Ribossomal RNA Processing 44) são responsáveis pela atividade catalítica e as demais subunidades envolvidas no direcionamento e seleção de transcritos para processamento ou degradação. O Trypanosoma rangeli, juntamente com outros tripanosomatídeos, apresentam deficiência no sistema de regulação de fatores de transcrição, portanto o mecanismo de regulação da expressão gênica nestes organismos ocorre predominantemente a níveis pós-transcricionais através da regulação dos níveis de RNA, sendo esses níveis regulados a partir da degradação dos transcritos. No presente estudo realizamos a caracterização de três subunidades do complexo exossoma de T. rangeli. A subunidade RRP6 de T. rangeli possui uma janela aberta de leitura de 2.133 pares de base que codifica para um polipeptídio de 710 aminoácidos (± 78,7 kDa). Foram identificados os domínios conservados PMC2NT, DEDD-Y e HRDC, que também se encontram presentes em outros organismos em que o complexo exossoma de RNA está caracterizado. Foi realizado teste de expressão heteróloga desta proteína em E. coli BL21 CodonPlus, porém sem sucesso de isolamento. A subunidade RRP44 de T. rangeli possui uma janela aberta de leitura de 3012 pares de base que codifica para um polipeptídio de 1003 aminoácidos (± 113,2 kDa). Os domínios conservados presentes nesta proteína são PIN, Exoribonuclease R e RNB. Foi realizada a expressão heteróloga desta proteína em E. coli BL21 CodonPlus, porém não tivemos sucesso do rendimento da proteína para o posterior etapa de purificação. A subunidade EAP3 (Exosome Associated Protein 3) possui uma janela aberta de leitura de 547 pares de base que codifica para um polipeptídio de 181 aminoácidos (± 20 kDa) e está descrita como uma proteína associada ao exossoma de RNA formando um heterodímero com a subunidade RRP6. Em ensaios de Western blot utilizando o antissoro produzido essa interação in vivo foi comprovada, uma vez que a ligação do anticorpo ocorreu na banda de ±150 kDa. As sequências do complexo exossoma de RNA apresentam porcentagens de identidade altas, demonstrando ser uma maquinaria conservada entre os tripanosomatídeos, porém com sua funcionalidade ainda desconhecida.

Abstract : RNA exosome complex is a multiprotein complex composed of 12 subunits, which has 3 '? 5' catalytic activity and is involved in the processing and degradation of several types of RNAs. The subunits RRP6 (Ribosomal RNA Processing 6) and RRP44 (Ribosomal RNA Processing 44) are responsible for the catalytic activity, and the other subunits are involved in the targeting and selection of transcripts for processing or degradation. Trypanosoma rangeli, together with other trypanosomatids, are deficient in the regulation system of transcription factors, so the regulation mechanism of gene expression in these organisms occurs exclusively at post-transcriptional levels through of RNA levels regulation, these levels being regulated from the degradation of the transcripts. In the present study we performed the characterization of subunits of the T. rangeli exosome complex. The RRP6 subunit of T. rangeli has an open reading frame of 2,133 base pairs coding for a polypeptide of 710 amino acids (± 78.7 kDa). The conserved domains identified was PMC2NT, DEDD-Y and HRDC, which are also present in other organisms in which the RNA exosome complex is characterized, have been identified. A heterologous expression test of this protein was performed in E. coli BL21 CodonPlus, but with no success of isolation. The RRP44 subunit of T. rangeli has an open reading frame of 3012 base pairs encoding a polypeptide of 1003 amino acids (± 113.2 kDa). The conserved domains present in this protein are PIN, Exoribonuclease R and RNB. The heterologous expression of this protein was performed in E. coli BL21 Codon Plus, but we did not have a success of protein yield for the subsequent protein purification process. The EAP3 (Exosome Associated Protein 3) subunit has an open reading frame of 547 base pairs encoding a polypeptide of 181 amino acid (± 20 kDa) and is described as a protein associated with the RNA exosome forming a heterodimer with the subunit RRP6. In Western Blotting assays using the antiserum produced from purified protein this in vivo interaction was proven, since antibody binding occurred in the ~ 150 kDa band, which corresponds to the sum of the proteins. The sequences of the RNA exosome complex have high identity percentages, demonstrating that it is a conserved machinery among the trypanosomatids, but with its still unknown functionality.

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Almairac, Fabien. "Plasticité des cellules tumorales de glioblastomes : inter-conversion d’un phénotype différencié et souche en fonction du microenvironnement." Thesis, Nice, 2016. http://www.theses.fr/2016NICE4045/document.

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L’objectif de ce travail était de démontrer que les cellules de glioblastomes sont capables de se différencier et de se dédifférencier en fonction de leur environnement, d’explorer les mécanismes biologiques qui sous-tendent ces transitions, et d’évaluer in vivo les capacités de différenciation à distance des CIG par les CIG-miR-302-367 via la sécrétion de microvésicules. A partir de plusieurs glioblastomes fraichement réséqués, nous avons caractérisé les cellules tumorales sur le plan phénotypique et fonctionnel pour l’état souche et différencié. Nous avons extraits et analysés les microvésicules des milieux de culture de 2 lignées de CIG-miR-302-367. Selon les principes de la thérapie cellulaire, des co-injections de CIG+CIG-miR-302-367 ont été réalisées dans le cerveau des souris. La majorité des cellules tumorales avaient un phénotype et étaient fonctionnellement différenciées. Après 48 heures de culture en milieu EGF, elles acquéraient les propriétés souches phénotypiques et fonctionnelles. Ce processus de dédifférenciation était réversible en 4 jours de culture en milieu sérum et inhibé par l’adjonction dans le milieu EGF d’un anti-EGFR (cétuximab), suggérant un rôle primordial de la voie EGF/EGFR/ERK. Les microvésicules produites par les CIG-miR-302-367 ont permis une baisse significative de la tumorigénicité des CIG in vivo, et une augmentation de la survie des souris. Le concept de plasticité cellulaire remet en cause les dogmes établis sur la hiérarchie tumorale unidirectionnelle. La déplétion tumorale en CIG, en les forçant à se différencier, est une stratégie thérapeutique innovante, qui peut s’envisager par une approche de thérapie cellulaire
There is great interest but little understanding in how cancer stem cells arise. Here we show that tumor cells exhibiting stem-like properties and expression of stemness(CD133) and pluripotency markers (SOX2, NANOG, OCT4), can arise from differentiated tumor cells that are isolated from human glioblastomas. These cells could transit from a more differentiated state that cannot self-renew to a self-renewing stem-like state upon EGF/EGFR signaling. This dedifferentiation process induced expression of pluripotency markers, and restored clonal and tumorigenic properties as well as resistance to temozolomide, the chemotherapy of reference. EGF/EGFR signaling including ERK activation was crucial for this cellular reprogramming. Interestingly, expression of pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating that the cells have the capacity to change and reprogram before the cell division starts. Our findings support a model of tumor homeostasis in which tumor cells driven by environmental cues such as EGF, can spontaneously acquire stem-like properties contributing thus to the enrichment in tumor propagating cells

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Chivet, Mathilde. "Exosomes neuronaux : rôle dans le passage intercellulaire de protéines et d'ARN." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00905660.

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Les exosomes sont des vésicules d'origine endocytaire sécrétées par les cellules dans leur environnement après fusion des endosomes multivésiculés avec la membrane plasmique. Ils représentent un nouveau moyen de communication cellulaire par le transfert intercellulaire de protéines, de lipides et d'ARN. Dans le laboratoire, nous nous intéressons aux rôles que pourraient jouer les exosomes neuronaux dans le système nerveux central. Nous avons montré que les neurones matures sécrètent des exosomes. Nous avons mis en évidence que cette sécrétion est directement reliée à l'activité synaptique glutamatergique et à une entrée de Ca2+. Nous avons également découvert que la partie C-terminale de la chaîne lourde de la toxine du tétanos peut être sécrétée par voie exosomale. Nous avons observé que les exosomes la contenant sont repris par des neurones en culture. Un tel cargo semble d'ailleurs influencer le devenir des exosomes. De plus, pour étudier la recapture des exosomes, nous avons utilisé des exosomes de cellules N2a exprimant la tétraspanine CD63 fusionnée à la GFP. En incubant des neurones d'hippocampe avec des exosomes GFP-CD63, nous sommes parvenus à démontrer qu'ils étaient endocytés par les neurones receveurs. Cependant, bien que les exosomes semblent avoir été internalisés, nos résultats suggèrent que leur trafic serait indépendant de la voie endocytaire classique. Enfin, nous nous sommes intéressé au contenu en ARN des exosomes de N2a et de neurones. Nous avons démontré qu'ils contenaient majoritairement des ARN courts (≤ 200 nucléotides) parmi lesquels, les microARN 132 et 138. Les microARN sont de puissants régulateurs de l'expression génique. Leur transfert, via les exosomes, représenterait une nouvelle voie de régulation très fine et avec un impact conséquent sur le fonctionnement du système nerveux. Les exosomes neuronaux pourraient donc jouer un rôle dans la physiologie normale de la synapse, en permettant l'échange d'ARN et de récepteurs aux neurotransmetteurs entre neurones. Ils pourraient également être impliqués dans la propagation de protéines pathogènes comme la toxine du tétanos et la propagation de certaines maladies neurodégénératives comme Alzheimer et Creutzfeldt-Jacob. L'ensemble de nos résultats suggère que les exosomes joueraient un rôle-clé dans le système nerveux central, de par leur implication dans des processus physiologiques et pathologiques.

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Santasusagna, Canal Sandra. "Exosomes i microRNAs en biòpsia líquida com a biomarcadors pronòstic del càncer de colon." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/482165.

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El càncer de colon (CC) és el tercer tipus de càncer més freqüent a nivell mundial i representa la segona causa de mort per càncer en homes i la tercera en dones. La majoria dels CC tenen un origen esporàdic a partir d’un pòlip benigne que progressa a un adenocarcinoma maligne degut a l’acumulació successiva de determinats canvis genètics i epigenètics. El principal factor pronòstic per la recaiguda i la supervivència en el CC és l’estadiatge de la malaltia, essent l’estadi III el que està associat a major risc de recaiguda i pitjor supervivència. El tractament més adequat en funció de les característiques clinicopatològiques de cada pacient és una qüestió controvertida i en constant evolució, sobretot pels pacients en estadi II. Per això actualment està adquirint gran importància l’estudi de nous biomarcadors pronòstic, com els exosomes i els RNAs no codificants petits (microRNAs), mitjançant biòpsia líquida i obtinguts a partir de les venes més pròximes al tumor, per tal de dissenyar un tractament personalitzat més eficient. Els exosomes són vesícules extracel·lulars (30-100nm) que poden afavorir el desenvolupament tumoral mitjançant la transferència de molècules empaquetades en el seu interior, com microRNAs i proteïnes, a les cèl·lules normals receptores. S’ha descrit que més del 40% del contingut exosomal correspon a microRNAs, l’expressió anòmala dels quals s’ha relacionat clàssicament amb el procés tumoral. El treball presentat ens ha permès valorar la utilitat dels exosomes i els microRNAs com a biomarcadors pronòstic en el CC. En el primer estudi amb mostres de pacients de CC, s’ha obtingut que alts nivells de miR-21 en la vena que drena directament el tumor primari, la vena mesentèrica (VM), s’associen a un pitjor pronòstic en termes de supervivència lliure de malaltia. Aquests resultats s’ampliaren al realitzar un array amb 754 microRNAs en VM i vena perifèrica (VP) de pacients quirúrgics de CC d’estadis I-III amb el qual s’obtingué un perfil de 4 microRNAs associats a valor pronòstic: let-7g, miR-15b, miR-155 i miR-328. Alts nivells de let-7g, miR-15b, miR-155 i miR-328, en VM s’associen a pitjor pronòstic en termes de temps a la recaiguda, mentre que, de forma interessant, s’obtenen resultats inversos a l’analitzar tals microRNAs en VP. Aquests resultats suggereixen que el tumor primari de colon allibera altes concentracions de microRNAs i exosomes a través de la VM i aquests nivells queden relativament diluïts en la circulació perifèrica. Aquest fet destaca la importància d’obtenir marcadors sanguinis abans de colonitzar l’òrgan diana. A continuació es van aïllar i caracteritzar exosomes de VM i VP dels pacients de la sèrie estudiada. Es va confirmar la presència de let-7g, miR-15b, miR-155 i miR- 328 en el seu interior mitjançant PCR quantitativa a temps real. El subgrup de pacients que va desenvolupar metàstasi presenta alts nivells de miR-328 exosomal en la VM. En base a aquests resultats previs, es procedeix a indagar l'efecte de miR-328 sobre els seus gens diana putatius per aprofundir en el mecanisme molecular que exerceix miR-328 durant el procés carcinogènic del CC. S'ha pogut comprovar que miR-328 inhibeix directament SLC2A1 / GLUT1 en dues línies cel·lulars de CC, LOVO i SW480, suggerint que miR-328 podria ser un element clau per modular les característiques metabòliques del microambient i afavorir la formació del nínxol pre-metastàsic, probablement participant en el desenvolupament de l'efecte Warburg. Finalment es va realitzar un anàlisi proteòmic dels exosomes de VM i VP dels pacients de la sèrie estudiada. Els resultats obtinguts mostren que els pacients recaiguts presenten alts nivells de la proteïna ECM1 exosomal en la VM i aquests s’associen a valor pronòstic.
Colon cancer (CC) is the third most common cancer worldwide. The main prognostic factor for relapse and survival is disease stage, and patients with stage III disease have a higher risk of relapse than those with stage II. Surgery is the standard treatment for stage I to III, and adjuvant treatment has been shown to be effective in stage III but less so in stage II. Consequently, the study of new prognostic and predictive biomarkers, such as exosomes and small non-coding RNAs, such as microRNAs (miRNAs), obtained from the tumor-draining vein can provide a useful tool for selecting treatment and improving outcome in these patients. In the present study, we have examined the role of exosomes and miRNAs as prognostic markers in CC. In a previous study, we examined the expression levels of the common oncogenic miRNA miR-21 in plasma samples from the tumor-draining mesenteric vein (MV) of CC patients and found that those with high expression had worse prognosis than those with low expression. In order to further explore this finding, we profiled the expression of 754 miRNAs in plasma samples taken from the MV and peripheral vein (PV) of surgically resected stage I-III CC patients. We found that let-7g, miR-15b, miR-155 and miR-328 in MV plasma were associated with shorter time to relapse. Interestingly, inverse results were obtained when analyzing the same miRNAs in PV plasma, highlighting the importance of obtaining blood biomarkers before they colonize the target organ. Next, we isolated and characterized exosomes from MV and PV and confirmed the presence of let-7g, miR-15b, miR-155 and miR-328 in exosomes. The subgroup of patients who developed metastases had high levels of exosomal miR-328 in MV. Based on these results, we proceeded to investigate the effect of miR-328 on its putative target genes during the development of CC. We found that miR-328 directly inhibits SLC2A1/GLUT1 in the LOVO and SW480 CC cell lines, suggesting that miR-328 could play a key role in modulating the metabolic characteristics of the microenvironment to induce the formation of the premetastatic niche. Finally, we performed a high throughput proteomic analysis in exosomes isolated from MV and PV from the same cohort of patients and found that relapsed patients showed high levels of ECM1 exosomal protein in the MV, which was associated with poor prognosis.

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Landmann, Emelie Maximiliane [Verfasser], and Malte [Akademischer Betreuer] Buchholz. "Rolle von Exosomen-lokalisierter RNAs in der Interaktion zwischen Tumorzellen und Makrophagen im Pankreaskarzinom / Emelie Maximiliane Landmann ; Betreuer: Malte Buchholz." Marburg : Philipps-Universität Marburg, 2019. http://d-nb.info/1189314916/34.

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Le, Gall Laura. "Secretion of neurotoxic vesicles by muscle cells of ALS patients." Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS210.pdf.

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A ce jour, les causes de la sclérose latérale amyotrophique (SLA) ne sont pas connues et il n’existe aucun remède. L’implication du muscle dans la pathogénèse de la SLA est toujours controversée, néanmoins il a été démontré qu’il existe une atteinte axonale distale primaire à l’apparition des premiers symptômes de la SLA. De plus, plusieurs données de la littérature suggèrent une perturbation de la voie de biogénèse et de sécrétion des exosomes chez certains patients atteints de forme mono-génique de la SLA. Sachant que le muscle squelettique peut être une source d’exosomes, nous émettons l’hypothèse que le muscle pourrait contribuer à la toxicité de l’environnement des motoneurones, à leur dégénérescence et donc à la pathogénèse de la SLA. J’ai donc étudié au cours de ma thèse la sécrétion d’exosomes par les cellules musculaires de patients sporadiques SLA et leurs rôles dans la SLA. Dans un premier temps, nous avons observé l’existence d’une signature associée à la SLA dans l’ensemble des cellules musculaires analysées, avec une accumulation et sursécrétion d’exosomes. Ces exosomes musculaires de patients SLA étaient toxiques pour les motoneurones dérivés d’iPSCs, induisant une réduction du nombre de branchements, une diminution de la longueur des neurites et une mort des motoneurones. Nous avons ensuite observé que les exosomes ALS contenaient la protéine FUS et d’autres protéines impliquées dans le transport et la maturation des RNAs. Or, le traitement des motoneurones avec des exosomes musculaire SLA induisait une accumulation de RNA dans leurs noyaux. Enfin, nous avons observé que la toxicité des exosomes SLA était dépendante du niveau d’expression de FUS de la cellule réceptrice. En effet, la toxicité des exosomes SLA était exacerbée lorsque FUS était surexprimé dans la cellule réceptrice et était diminuée lorsque l’expression de FUS était éteinte. Or les motoneurones présentent un niveau d’expression de FUS supérieur aux cellules musculaires, suggérant leur sensibilité accrue aux exosomes musculaires SLA. Ensemble ces données suggèrent une implication du muscle dans la dégénérescence des neurones moteurs de patients SLA
To date, amyotrophic lateral sclerosis (ALS) remain incurable and the causes for motor neuron death are unknown. The primary involvement of skeletal muscle in ALS pathogenesis is still controversial. Several studies suggested that a distal axon degeneration occurs prior to the onset of ALS clinical symptoms. Moreover, there are growing evidences for a disruption of exosomes biogenesis and secretion pathways in genetic forms of ALS. Knowing that the skeletal muscle can be a source of exosomes, we hypothesised that ALS skeletal muscle could contribute to the toxic environment of motor neurons and thus participate in ALS pathogenesis through their secretion of exosomes. During my PhD, I investigated the secretion of exosomes by muscle cells from sporadic ALS patients and their role in motor neuron death. First, we show that muscle cells present a consistent signature across ALS patients with an accumulation and over-secretion of exosomes. Second, ALS muscle exosomes are toxic toward healthy human iPSCs motor neurons by inducing shorter, reduced branching neurites and cell death. Third, we observed that ALS muscle exosomes contain FUS protein and are enriched in proteins involved in RNA maturation and transport. Fourth, ALS muscle exosomes induced a disruption in RNA transport in healthy human motor neuron. Fifth, the exosome toxicity is dependent on FUS expression level in the recipient cells, as an over-expression of FUS in the recipient cells exacerbated the ALS exosome toxicity while an inhibition of FUS expression decreased their toxic effect. The greater sensitivity of motor neurons to ALS muscle exosomes might thus be explained by their higher expression level of FUS compare to healthy muscle cells. Altogether, these results suggest that ALS muscle exosomes could contribute to the degeneration of motor neuron in ALS patients

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Büttner, Katharina. "Part I: Structural framework for the mechanism of archaeal exosomes in RNA processing; Part II: Structural insights into DNA duplex separation by the archaeal superfamily 2 helicase Hel308." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-71973.

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Ruiz, Martinez Marc. "Estudio del gen YKT6 y miRNAs asociados en la liberación de exosomas en pacientes quirúrgicos de cáncer de pulmón." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/401865.

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En los últimos años los exosomas han emergido como importantes mediadores en el desarrollo de cáncer, demostrándose su utilidad como biomarcadores para diagnóstico y pronóstico de pacientes oncológicos. Los exosomas son vesículas extracelulares de entre 30-100nm de diámetro de origen endosomal que pueden favorecer diferentes procesos importantes en el progreso tumoral, como la modulación del microambiente tumoral, inmunosupresión y preparación del nicho premetastático. El estudio de la biogénesis y liberación de los exosomas se ha centrado principalmente en los mecanismos ESCRT y de proteínas Rab, postulándose también la importancia de las proteínas SNARE en estos procesos. YKT6 es una proteína SNARE que se ha descrito como necesaria para la liberación de exosomas en diferentes tipos celulares. Sin embargo el papel que podría tener en la liberación de exosomas en cáncer de pulmón no se había investigado. El trabajo realizado nos ha permitido investigar la importancia de YKT6 en el proceso de liberación de exosomas en líneas celulares, sus mecanismos de regulación mediante microRNAs (miRNAs), así como también evaluar el posible papel pronóstico en pacientes de cáncer de pulmón de célula no pequeña. En el estudio in vitro con la línea celular A549 de cáncer de pulmón observamos que la inhibición de YKT6 producía una disminución drástica en la liberación de exosomas. Además, identificamos y validamos miR- 34a, miR-134, miR-135a y miR-135b como miRNAs capaces de regular la expresión del ARNm de YKT6. El efecto de la sobreexpresión de miR-134 y miR-135b, los miRNAs que más efectivamente disminuían la expresión de YKT6 también provocaron una disminución en la liberación de exosomas en las células A549. En los pacientes de cáncer de pulmón de célula no pequeña observamos que la expresión de YKT6 estaba infraexpresada y que en contraste la expresión de los miRNAs miR-134 y miR-135b se encontraban sobrexpresados en tejido tumoral respecto al tejido normal. Aunque en el tejido tumoral no se pudo apreciar ninguna correlación negativa entre los miRNAs y la expresión de YKT6, en el tejido normal si que observamos la correlación negativa significativa entre miR-135b y la expresión de YKT6. El análisis de la expresión de YKT6 en las muestras tumorales de los pacientes resultó en la clasificación de los pacientes según niveles de expresión alto o bajo de YKT6, observándose un peor pronóstico en el grupo de pacientes con alta expresión en términos de una menor supervivencia global y supervivencia libre de enfermedad. Estos resultados indican que el estudio de los procesos de liberación de exosomas y sus posibles mecanismos de liberación, en este caso YKT6 a través de miRNAs, pueden ser de utilidad en cáncer.
Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non- small cell lung cancer (NSCLC) has not been examined. This study has allowed us to investigate the importance of YKT6 in the process of releasing exosomes in cell lines, its mechanisms of regulation by microRNAs (miRNAs), as well as to evaluate the possible prognostic role in patients with non-small cell lung cancer . In the in vitro study with the lung cancer cell line A549, we observed that the inhibition of YKT6 produced a drastic decrease in the release of exosomes. In addition, we identified and validated miR-34a, miR-134, miR-135a and miR-135b as miRNAs capable of regulating the expression of YKT6 mRNA. The effect of overexpression of miR-134 and miR-135b, miRNAs that most effectively decreased YKT6 expression also led to a decrease in the release of exosomes in A549 cells. In non-small cell lung cancer patients we observed that the expression of YKT6 was underexpressed and that in contrast the expression of miRNAs miR-134 and miR-135b were overexpressed in tumor tissue compared to normal tissue. Although no negative correlation between miRNAs and YKT6 expression in the tumor tissue was observed, in the normal tissue, we observed a significant negative correlation between miR-135b and YKT6 expression. Analysis of YKT6 expression in patient tumor samples resulted in the classification of patients according to high or low expression levels of YKT6, with a worse prognosis in the group of patients with high expression in terms of lower overall survival and disease-free survival. These results indicate that the study of the exosome release processes and their possible release mechanisms, in this case YKT6 through miRNAs, may be useful in cancer.

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44

Sutaria, Dhruvitkumar S. "INVESTIGATION OF DIFFERENTIALLY EXPRESSED NONCODING RNAS IN PANCREATIC DUCTAL ADENOCARCINOMA." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480550158159039.

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45

Tseng, Yi-Ting, and 曾依婷. "RNA degradation machine: molecular machanism by which the E. coli PNPase-exosome finds its RNA substrates." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/78413313529809634020.

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碩士
國立陽明大學
生命科學暨基因體科學研究所
98
In eukaryotes and archaea, the main protein complexes involved in 3¢-to-5¢ RNA degradation and RNA processing are exosomes that have similarities in structure and domain organization to the trimeric bacterial PNPase complex. In E. coli, PNPase has been shown to physically interact with RNA helicase B (RhlB), and the degradation of double-stranded RNA by PNPase requires RhlB. Here, we investigate the biological significance of the interaction between RhlB and trimeric PNPase. Our previous microarray data showed that a group of cys transcripts involved in the organic sulfate transport and metabolism pathway is up regulated when the interaction between PNPase and RhlB is disrupted. We used different deletion strains to check the stability of these transcripts by Northern blot analysis and found that the stability of these transcripts remained unchanged. We also confirmed this result under conditions under which interactions between PNPase and RhlB are weakened. In the pathway of organic sulfate transport and metabolism, a transcriptional activator called CysB is involved in the regulation of the cys operon. We checked the stability of the cysB transcript and found that the deletion of the PNPase-based exosome or disruption of the interaction between PNPase and RhlB stabilized cysB mRNA. Taken together, our research suggests that the stability of cysB is regulated by the PNPase-based exosome, which is dependent on the interaction of PNPase and RhlB, and that the increase of other cys operons is a secondary effect of CysB. Our study implies that the interaction between RhlB and PNPase is important for the regulation of sulfur metabolism in E. coli and provides a candidate for studying the function of the PNPase-based exosome.

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46

Pefanis, Evangelos. "RNA Exosome Regulated Antisense and Divergent Noncoding RNA Facilitate AID Targeting Throughout the B Cell Genome." Thesis, 2015. https://doi.org/10.7916/D8B56HH2.

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Vertebrate immune systems are armed with the ability to generate highly specific immune responses capable of responding to nearly any foreign molecular threat. One of the major mediators of this response is immunoglobulins (Igs) produced by B lymphocytes. The specificity of individual Igs is created through a tightly orchestrated series of somatic DNA manipulations at Ig encoding loci resulting in functional gene rearrangements and nucleotide substitutions. These events serve to create a pool of naive B cells expressing Igs with distinct specificities, capable of expansion in response to antigen specific selection. Affinity of Ig towards antigen is enhanced through nucleotide substitutions introduced at the antigen binding variable region gene segments through the enzyme activation induced cytidine deaminase (AID) during the process of somatic hypermutation (SHM). AID also generates point mutations within noncoding DNA segments of the Ig heavy chain locus that are processed into double strand breaks leading to constant region isotype switching during class switch recombination (CSR). The Ig diversification processes of SHM and CSR critically depend upon transcriptional activation of the relevant DNA segments. Transcription is thought to facilitate single strand DNA substrate recognition by AID during unwinding of the DNA duplex. The 3'-5' exoribonuclease RNA exosome serves as a transcription dependent cofactor of AID. RNA exosome is comprised of multiple structurally integral core subunits and associated nuclease subunits. In this work, RNA exosome core subunit Exosc3 and nuclease Exosc10 have been targeted for conditional mutagenesis and loss of function analysis in mouse cells. RNA exosome deficient B cells were significantly impaired in AID dependent SHM and CSR Ig diversification processes. Transcriptome analyses revealed a striking accumulation of promoter proximal antisense divergent noncoding transcripts (xTSS-RNA) at a subset of genes upon loss of RNA exosome function. xTSS-RNAs mark regions of chromatin containing RNA exosome activity. Multiple known AID target sites including IgH and Myc were observed to express xTSS-RNA. Furthermore, genomic sites of recurrent AID dependent chromosomal translocations were enriched for xTSS-RNA. In addition to promoter proximal xTSS-RNA, cryptic intragenic antisense noncoding transcripts were found to accumulate at many genomic loci. In fact, multiple translocation hotspots precisely overlap regions of RNA exosome sensitive antisense transcription. AID targeted divergently transcribed promoters containing RNA exosome substrates possessed greater amounts of RNA:DNA hybrids, indicative of frequent transcriptional arrest. Lastly, RNA exosome deficient transcriptomes have revealed a substantial number of novel long intergenic noncoding RNAs and enhancer RNAs, indicating a hidden layer of cellular transcriptional activity. A model of AID targeting utilizing transcriptional arrest is becoming increasingly apparent. Transcribed chromatin prone to undergo transcriptional arrest, such as Ig loci or xTSS-RNA expressing regions, frequently undergoes premature transcription termination coupled to RNA exosome mediated degradation of the nascent transcript. This process results in the creation of AID substrates and serves to stabilize its association with chromatin through multiple interactions involving RNA exosome and transcription complex subunits.

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47

Yang, Jie Eune. "Exploration of a novel non-lytic viral transmission mechanism utilized by a non-enveloped positive-sense RNA virus." Thesis, 2018. https://hdl.handle.net/2144/29955.

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While enteroviruses, including poliovirus, are conventionally released upon cell lysis, recent studies show that phosphatidylserine-enriched infectious extracellular vesicles (IEVs) shed by infected cells can transport clusters of enteroviruses from cell to cell, resulting in increased infectivity. Combining structural and biochemical analyses, we focused on IEVs shed from poliovirus-infected cells, a classical prototype for studying enteroviruses. Transmission cryo-electron microscopy, cryo-electron tomography and computational reconstruction, present the first three-dimensional structures of well-preserved IEVs and purified exosomes. We observed that single-membraned IEVs present a wide size range in diameter. Clusters of virions can be either densely packed within a protein-coated irregularly shaped IEV, or concentrated at one or both ends of an IEV, forming a polar structure. In addition to virions, IEVs often contain internal vesicles, “ramen-noodle”-like structures with strong density, and partially assembled virion-like structures. Viral replication complex components, including viral proteins polymerase 3D, 3CD, 3A, 3AB, 2BC, 2C and (+) and (-) stranded RNAs were detected in IEVs. Furthermore, (-) stranded RNA templates are protected by the IEVs, not packed in viral capsids. The transported viral replication components (viral proteins and RNAs) and virions within IEVs initiate a stronger and faster viral replication in recipient cells than free virions. Both cryo-electron tomographic and mass spectrometry data also showed that virions and “ramen-noodle”-like structures were also observed in purified CD9 positive exosomes from poliovirus-infected cells. Viral protein 3AB, detected on the membrane of IEVs, can invaginate membranous structures to engulf large proteins into a closed lumen. Our study demonstrates that IEVs can transport viral replication complex components to initiate a rapid onset of viral replication, as part of a novel viral transmission mechanism. Viral protein 3AB may contribute to forming IEVs throughout the infection.
2019-06-12T00:00:00Z

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48

Wun, Sheng-Fa, and 温勝發. "Exosome-carried YTH methyladenosine RNA binding domain family 2 (YTHDF2) protein Induced Inflammatory Signaling in nonalcoholic fatty liver disease." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/2jen74.

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碩士
國立陽明大學
臨床醫學研究所
107
Hepatocyte cellular dysfunction and pro-inflammtion by lipid induced release of exosomes(EXO) from hepatocytes, which activate an inflammatory phenotype are characteristics of nonalcoholic steatohepatitis (NASH).Exosomes are extracellular vesicles responsible for communication between cells. However, the regulatory mechanism of YTH domain methyladenosine RNA binding family 2 (YTHDF2) protein is not understand. In this study, it was shown that incubation with lysophosphatidylcholine (LPC), a central player for hepatic fat accumulation and inflammation, resulted in dose-dependent increase in fatty acid accumulation assessed by Oil Red O staining. We have found that treatment of LPC-induced lipotoxicity promoted the expression of m6A reader YTHDF2. Interestingly, the LPC treatment at the concentration of 20 µM shown to significantly increase nanoparticles secretion compared vehicle control based on nanoparticle tracking analysis (NTA). Moreover, these isolated exosomes were positively stained CD63 and YTHDF2 by using super-resolution fluorescence microscopy and co-localization is confirmed. We investigated the effect of the exosomes-carried YTHDF2 protein derived from overexpression YTHDF2 of human hepatocytes (HepG2) on human hepatocytes in vitro. In vitro data showed that EXO could be uptake and exert paracrine effect in the human hepatocytes exposing to different doses. Quantitative PCR detection showed that EXO treatment significantly induced the upregulated expression of pro-inflammatory genes in the human hepatocytes. Intriguingly, western blotting detection demonstrated that YTHDF2 overexpression recovered the phosphorylation levels of p38, suggesting the involvement of the YTHDF2-mediated p38 signaling pathway in injured livers. In conclusion, our findings demonstrate that EXO derived from LPC-induced human hepatocytes aggravate nonalcoholic fatty liver disease and the mechanism may involve an EXO carried YTHDF2 protein-triggered p38 Inflammatory Signaling pathway.

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49

Hartung, Sophia [Verfasser]. "Analyzing protein-nucleic acid complexes using hybrid methods : I. the DNA damage checkpoint protein DisA; II. structural biochemistry of RNA turnover by the exosome / Sophia Hartung." 2008. http://d-nb.info/994570406/34.

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50

Gendron, Louis. "Adaptation de la levure à la suite des perturbations du mécanisme de contrôle de qualité de l'ARN." Thèse, 2019. http://hdl.handle.net/1866/23638.

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The life-cycle of RNA is determined by several processing steps, which allow the cell to export and translate a coding transcript. The cell has developed an astonishingly complex mechanism to ensure the integrity of RNA processing steps. The quality control mechanism of RNA balances the biosynthesis and degradation of various transcripts, adding another layer of gene regulation to the complex system of gene expression. The exosome is a central piece of the RNA quality control mechanism as it degrades many of the aberrant or non-functional RNAs in the nucleus and the cytoplasm. This project characterizes and highlight a response to mutation of components from the RNA quality control mechanism in Saccharomyces cerevisiae. These perturbations include functional components of the exosome (Csl4 and Dis3), a cofactor of the nuclear exosome (Rrp6), an essential protein for pre-rRNA processing (Enp1) and a component of RNA export machinery (Srm1). Here, I present bioinformatics approaches to characterize the cellular response at a level of transcript expression and polyadenylation size. The stress response embedded in the gene expression profile is highly similar between the mutants. This work suggests a generic response to a failure in different components of the RNA quality control machinery.
Le cycle de vie des ARN est déterminé par différentes étapes permettant à la cellule d’exporter et de traduire un transcrit codant. La cellule a développé un mécanisme incroyablement complexe pour s’assurer de l’intégrité des étapes de maturation de l’ARN. Le mécanisme de contrôle de qualité balance la biosynthèse et la dégradation de différents transcrits, ce qui ajout un niveau de régulation au système de l’expression génique. L’exosome est une pièce centrale du mécanisme de contrôle de qualité de l’ARN alors qu’elle dégrade une grande partie des transcrits aberrants ou non-fonctionnels dans le noyau et le cytoplasme. Ce projet caractérise et souligne la réponse cellulaire à la suite de la mutation de composantes du mécanisme de contrôle de qualité de l’ARN chez Saccharomyces cerevisiae. Ces perturbations comportent des composantes fonctionnelles du complexe de l’exosome (Csl4 et Dis3), un cofacteur de l’exosome nucléaire (Rrp6), une protéine essentielle pour la maturation des pré-ARNr (Enp1) et une composante de la machinerie d’export de l’ARN (Srm1). Ici, je présente des approches bio-informatiques pour caractériser la réponse cellulaire au niveau de l’expression des transcrits et de la taille des segments polyadénylés. La réponse au stress cellulaire intégré dans le profil d’expression du génome est très similaire entre les mutants. Ce travail suggère une réponse générique à la suite de la perturbation de différentes composantes du mécanisme de contrôle de qualité de l’ARN.

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Dissertations / Theses on the topic 'RNA exosome'

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