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Janzen, Timothy William, and University of Lethbridge Faculty of Arts and Science. "Subunit interactions within box C/D sRNPs." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, c2010, 2010. http://hdl.handle.net/10133/2547.
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Box C/D small ribonucleoproteins (box C/D sRNPs) are responsible for the 2’-O-methylation required for the complete maturation of precursor rRNA. Archaeal box C/D sRNPs, like eucarya, are composed of four components: a guide RNA (box C/D sRNA), an RNA binding protein (L7ae), a 2’-O-methyltransferase (Fibrillarin) and a structural protein (Nop5). Here we develop several approaches for studying box C/D sRNP assembly. In particular, we have used pulldown and mobility shift assays to identify box C/D sRNP assembly intermediates (Nop5-aFib and L7ae-sR1). We have also demonstrated that isothermal titration calorimetry (ITC) can be utilized to quantitatively characterize the energetics of formation for the L7ae-sRNA assembly intermediate.xi, 98 leaves : ill. (some col.) ; 29 cm
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Malliahgari, Srinivas Reddy. "Characterization of a box C/D RNA from Haloferax volcanii /." Available to subscribers only, 2006. http://proquest.umi.com/pqdweb?did=1240690311&sid=3&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Thesis (M.S.)--Southern Illinois University Carbondale, 2006."Department of Molecular Biology, Microbiology and Biochemisty." Includes bibliographical references (leaves 59-61). Also available online.
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Bosmeny, Michael. "Structure / Function Relationship of Archaeal Box C/D and H/ACA Proteins." OpenSIUC, 2016. https://opensiuc.lib.siu.edu/theses/1901.
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Ribonucleoprotein complexes are responsible for some of the post-transcriptional modifications of RNA that occur within the cell, including 2'-O-methylation and pseudouridylation. These modifications contribute, among other things, to RNA folding, inhibition of degradation, and general cellular viability. In this study, we identify residues within the proteins of these complexes that are important to the functioning of the Box C/D and Box H/ACA complexes. Candidates were selected based on previous work and mutant versions of the proteins were introduced in-vivo. Assays were done to determine the functionality of the mutant complex. This work is divided into three parts, focused on the three proteins investigated. The first part is concerned with Nop5, a protein in the Box C/D RNP complex. Nop5 is known to interact with all other proteins and RNAs in the complex, and is believed to serve a primarily structural role, aligning the other components. Mutagenesis study of suspected significant amino acids in this protein showed that it is difficult to disrupt the operation of Nop5 with single changes, but is possible with more extensive mutation. The second part concerns Fibrillarin, the catalytic protein of the Box C/D ribonucleoprotein complex. Previous mutagenesis work identified several important amino acids involved with AdoMet transfer and complex formation. The methylation ability of these mutant complexes were further examined in this work by confirming that the same modification, or lack thereof, occurred at a second rRNA position. The final part of this work is about Nop10, part of the Box H/ACA complex. This work is only preliminary, but begins the process of testing suspected essential amino acids in the structure.
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Kaushik, Saakshi. "A STUDY TO UNDERSTAND THE DYNAMICS OF ARCHAEAL BOX C/D MEDIATED RNA MODIFICATION." OpenSIUC, 2015. https://opensiuc.lib.siu.edu/theses/1746.
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Stable cellular RNAs undergo a variety of post-transcriptional modifications, most of which are evolutionary conserved and found in functionally important regions of RNA. 2’-O-methylation of ribose sugars and conversion of uridine to pseudouridine by isomerization are the two most common modifications found in RNA in all the domains of life. 2’-O-methylation of the hydroxyl group of ribose is known to help in proper RNA folding, inhibit hydrolytic degradation and attack by nucleases on RNA. In Archaea, one of the ways this modification is known to be carried out is in an RNA-dependent manner by a Box C/D sRNP complex. This thesis sets to understand how exactly the RNA and proteins work together to bring about this modification. In the first part of this study, we aimed to investigate the role of Nop5, a protein component of the Box C/D sRNP whose function is not well understood. It was observed from previous work in our lab that aNop5p binds to single-stranded bulges and loops of some RNA. The binding seems to happen either by aNop5p alone or as a heterodimer with fibrillarin. This led us to hypothesize that Nop5 protein is responsible for binding to the target RNA to be methylated and bringing it to the assembling sRNP complex. To further investigate this idea, we identified two motifs in the C-terminal domain of the protein, the GAEK and ALFA motifs, mutated the motifs in the M. jannaschii versions of the protein and studied its significance in vitro using activity and binding assays. We have biochemically identified these motifs to be essential for the methylation activity of the sRNP complex and required to wedge open the guide strands for the target RNA to pair up. However, mutation of the motifs did not seem to change the way Nop5p binds to the bulges and loops of the RNA, inferring that these motifs are not essential for target identification and recruitment. The second part of the thesis is dedicated to the establishment and optimization of a pulldown technique using S1 RNA aptamer. This aptamer is inserted in a Box C/D guide RNA and used to purify the RNP complex formed in vivo using the strong affinity of the aptamer to streptavidin. The aptamer tag was successfully inserted into the 124mer form of sR-tMet of H. volcanii and the ability of the aptamer to bind to the streptavidin beads was tested. The roadblocks of the study, i.e. the non-specific binding and elution of the complex are currently being optimized.
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Pinzon, Restrepo Natalia. "Characterization of regulatory noncoding RNAs : the U1 small nuclear RNA and Cajal body-specific box C/D guide RNAs." Toulouse 3, 2011. http://thesesups.ups-tlse.fr/2458/.
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Les cellules contiennent de nombreux ARNs non-codants qui jouent un rôle essentiel dans toutes les étapes de l'expression génique. Nos travaux concernent la caractérisation de possibles nouvelles fonctions associés à deux classes d'ARNs non-codants : l'ARN U1 et les scaARNs. Le petit ARN nucléaire U1 forme une ribonucléoparticule (snRNP U1) qui joue un rôle clé dans l'épissage des ARN pré-messagers. De plus, l'ARN U1 semble intervenir dans d'autres étapes de la synthèse des ARNm fonctionnels. Nos travaux ont démontré qu'une fraction de l'ARN U1 interagit spécifiquement avec la protéine TAF15, dans un complexe qui diffère dans sa composition protéique de la particule d'épissage canonique Sm snRNP U1. Nous avons constaté qu'à la différence de la particule d'épissage, la snRNP U1-TAF15 est fortement attachée à la chromatine. De plus, suite à l'arrêt de la transcription par l'ARN polymérase II, la particule U1-TAF15 se relocalise dans la coiffe périnucléolaire, à l'opposé de la particule Sm snRNP U1 qui s'accumule alors dans les "speckles". Une étude protéomique de la particule U1-TAF15 nous a permis d'établir des hypothèses quant à sa fonction qui sont actuellement à l'étude dans le laboratoire. Récemment, une protéine essentielle à l'adressage des scaARNs (ARNs spécifiques des Corps de Cajal) dans les Corps de Cajal a été identifiée (Tycowski et al. , 2009; Venteicher et al. , 2009). Une analyse par séquençage à haut-débit de l'ensemble des petits ARNs associés à WDR79 a permis d'identifier de nombreux nouveaux scaARNs putatifs. Parmi eux, l'analyse de deux ARNs à boite C/D a défini des structures essentielles pour leur localisation dans les corps de Cajal. Des études plus approfondies devraient nous permettre de déterminer des éléments spécifiques nécessaires à l'adressage des scaARNs à boite C/D vers les corps de Cajal. Par ailleurs, nous avons commencé la caractérisation d'un nouveau scaARN qui ciblerait la modification d'un ARN de transfert. Alors que les ARN C/D connus chez les Eucaryotes participent à la biogenèse des ARN ribosomiques et des petits ARN nucléaires, nous avons identifié pour la première fois chez les Eucaryotes un ARN C/D qui ciblerait un ARN de transfertNoncoding regulatory RNAs (ncRNAs) are in the focus of current research, since they participate in nearly all cellular processes. To get further insights into the functional and structural complexity of ncRNAs, we studied human ncRNAs belonging to two classes of ncRNAs, the nucleoplasmic spliceosomal snRNAs and the nucleolar and Cajal body-specific box C/D 2'-O-methylation guide RNAs. The U1 snRNP is an evolutionarily conserved, abundant nucleoplasmic snRNP that plays a central role in pre-mRNA splicing. According to a recently emerging view, besides its constitutive role in splicing, the U1 snRNP has important regulatory functions in different steps of pre-mRNA production. We demonstrated that a fraction of the human U1 snRNA specifically associates with the nuclear RNA-binding protein TAF15 that is known to interact with a subpopulation of TFIID and RNA polymerase II complexes. The U1-TAF15 snRNP is structurally and functionally distinct from the well-characterized U1 spliceosomal snRNP and it tightly associates with chromatin. The function of U1-TAF15 snRNP remains unknown; it might contribute to the coupling of transcription and splicing. WDR79 (also called WRAP53) has been recently identified as an essential factor for targeting a subclass of box C/D and H/ACA modification guide RNAs, as well as telomerase H/ACA RNA, into the Cajal bodies. Accumulation of box C/D and H/ACA RNPs in Cajal bodies is essential for the biogenesis of functional spliceosomal snRNPs and telomere synthesis. Co-immunopurification of WDR79-associated human RNAs, followed by cDNA synthesis and deep sequencing identified a large number of novel Cajal body-specific RNAs. We are currently dissecting the cis-acting RNA element responsible for WDR79-binding and for targeting box C/D 2'-O-methylation guide RNPs into Cajal bodies. We have also identified a novel Cajal body-specific 2'-O-methylation guide RNA that is predicted to direct methylation of cytidine 34 at the Wobble position of tRNA-Met-CAT elongator. Interestingly, tRNA modification is a novel function for vertebrate box C/D scaRNPs
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Jana, Sujata. "STRUCTURAL AND FUNCTIONAL STUDIES OF ARCHAEAL BOX C/D GUIDE RNA AND ROLE OF A PUTATIVE HUMAN PSEUDOURIDINE SYNTHASE, PUS10 IN APOPTOSIS." OpenSIUC, 2017. https://opensiuc.lib.siu.edu/dissertations/1362.
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RNAs undergo different posttranscriptional chemical modifications, which affect their structural stability and functional diversity. RNA methylation is a very common type of post-transcriptional modification and is present in all domains of life: Archaea, Eukaryotes and Bacteria. Some of these methylations are catalyzed either by a RNA-protein complex or by stand-alone enzymes. The RNA-protein complex (Ribonucleoprotein complex) is comprised of a small RNA known as the guide RNA (Box C/D RNA) and core proteins (L7Ae, Nop5, and Fibrillarin). Box C/D RNAs contain conserved regions, called box C and box D near their 5’ and 3’ termini, respectively, and their imperfect copies called box C’ and box D’, internally. A short stretch of sequence between these Boxes are known as the guide/spacer regions, as the guide region helps in recruiting and positioning a specific target RNA for modification. Both in Archaea and Eukarya, box C and box D, as well as box C’ and box D’ together can form a structure called a Kink-turn (K-turn) that is characterized by a canonical Watson-Crick base-paired stem on one side, and a non-canonical stem on the other, separated by a 3-nucleotide loop. In Archaea box C’ and D’ can also form a K-loop, where the canonical stem of K-turn is replaced by a loop. Archaeal L7Ae binds first to the K-turn or K-loop and allows the recruitment of other proteins to form the complex. The presence of a unique box C/D RNA of Haloferax volcanii, called sR-tMet has been reported previously to guide the 2’-O-methylation of C34 in elongator pre-tRNAMet. Here we tried to characterize the structure-function relationship of this guide RNA under in vivo conditions. This RNA lacks a conventional K-turn or K-loop at its C’/D’ motif. We have created an H. volcanii strain that has a genomic deletion of sR-tMet. The sR-tMet gene is not essential for H. volcanii but this sR-tMet deleted strain lacks the 2’-O-methylation of C34 of its elongator tRNAMet. Unlike the close sR-tMet homologs (sR8 from Methanocaldococcus jannaschii and sR49 from Pyrococcus abyssi), the Box C’/D’ motif of sR-tMet is neither a K-turn nor a K-loop. The introduction of proper K-loop in the Box C’/D’ motif (sR-tMet with K-loop) abolished its Cm34 modification function in ΔsR-tMet strain. Direct interaction between L7Ae and the K-loop is not an absolute requirement for its function. However, disruption of the G/A and A/G pairing in Box C/D motif and Box D’ suggests the importance of these non-Watson crick base pairings in respect to sR-tMet’s function. Several other mutational studies have revealed that peculiar sR-tMet guide RNA from H. volcanii, behaves more like a Eukaryotic Box C/D RNA (where the K-loop is not required and presence of longer spacer length) than regular Archaeal one. Pseudouridine synthase 10 (Pus10) is the most recently identified Ψ synthase, found only in higher eukaryotes and Archaea. Archaeal Pus10 produces either tRNA Ψ55 or both tRNA Ψ54 and Ψ55 modifications. In Human, its Ψ synthase activity is not yet confirmed and interestingly it has been implicated in apoptosis. Herein for the first time we revealed that this putative RNA Ψ synthase protein, Human Pus10 (HuP10), translocates from the nucleus to the cytoplasm in TRAIL induced apoptosis. This nucleo-cytoplasmic movement of HuP10 occurs through the CRM1 mediated nuclear export pathway and Caspase 3 influences this movement. HuP10 also mediates crosstalk between the extrinsic and intrinsic pathways during TRAIL-induced apoptosis. Other than its involvement in apoptosis, we have also uncovered that HuP10 is involved in regulation of cell proliferation. Depletion (knockdown) of this protein in different cancer cell lines, promotes cell migration and anchorage-independent cell growth in the absence of any apoptotic stimulation.
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Singh, Sanjay K. "Functional and structural studies of an archaeal intron-encoded, cis-positioned box C/D guide RNA /." Available to subscribers only, 2006. http://proquest.umi.com/pqdweb?did=1140183781&sid=4&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Thesis (Ph.D.)--Southern Illinois University Carbondale, 2006."Department of Molecular Biology, Microbiology and Biochemisty." Includes bibliographical references (leaves 116-136). Also available online.
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Chatterjee, Kunal. "A TALE OF TWO METHYLATION MODIFICATIONS IN ARCHAEAL RNAs." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/806.
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In all the three domains of life, most RNAs undergo post transcriptional modifications both on the bases as well as the ribose sugars of the individual ribonucleotides. 2'-O-methylation of ribose sugars and isomerization of Uridines to Pseudouridines are two most predominant modifications in rRNAs and tRNAs across all domains of life. Besides 2'-O-methylation of ribose sugars, methylation of pseudouridine (Ø) at position 54 of tRNA, producing m1Ø, is a hallmark of many archaeal species but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m1Ø was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m1Ø minus phenotype of the ÄHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TØ-arm (17-mer) fragments as substrates, the sequential pathway of m1Ø54 formation in Archaea was reconstituted. The methylation reaction is AdoMet-dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TØ-loop. The presence of Ø55 allowed the efficient conversion of Ø54 to m1Ø54, whereas in the presence of C55 the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins. Another aim of this study was to investigate the mechanism of target RNA recruitment to a box C/D sRNP. From data obtained, we have made the following hypothesis- aNop5p, either alone or as a heterodimer with Fibrillarin, binds to single stranded bulges and loops of target RNA. This aNop5p bound target is then hybridized to an assembling guide sRNP complex containing the guide RNA and L7Ae or guide RNA, L7Ae and aNop5p. If the guide:target sequences are complementary to each other, they hybridize and the target nucleotide gets modified. We also think that post modification, the guide and target strands separate, the core proteins rearrange themselves on the guide RNA and then prime the guide RNA for next round of modification. Compared to the general archaeal populations, haloarchaea contain significantly fewer number of box C/D guide RNAs. In archaea, previous studies have underscored the importance of a symmetric assembly of the core proteins on the sRNA. This meant that if the core proteins were unable to bind to either the terminal box C/D or the internal box C'/D' motifs, the sRNP was not efficient to carry out the modification of the target RNA. Essentially the only two haloarchaeal box C/D sRNPs known before had a symmetric architecture. In this study we discovered the first naturally occurring asymmetric box C/D sRNP called sR-41 in Haloferax volcannii. The architecture of Haloferax volcanii sR-41 box C/D sRNP seems to be closer in conformation to eukaryal snoRNPs (eukaryal counterparts of archaeal sRNPs) in which the core proteins assemble asymmetrically on the RNA. Till date, no information regarding the catalytic mechanism of an asymmetrically arranged eukaryal box C/D snoRNPs are available, because of unavailability of any assembly systems or crystal structures. Hence, this archaeal sR-41 guide sRNP provides a unique opportunity to study mechanism of modification in an asymmetrically arranged box C/D sRNP molecule.
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Rothé, Benjamin. "Étude des processus de biogenèse des petites particules ribonucléoprotéiques nucléolaires à boîtes C/D (snoRNP C/D) chez la levure Saccharomyces cerevisiae : caractérisation fonctionnelle et structurale d'une machinerie dédiée à l'assemblage de ces RNP." Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0013/document.
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Les protéines de la famille L7Ae sont les constituants de nombreuses RNP essentielles. Chez les vertébrés, les particules snoRNP C/D et H/ACA sont impliquées dans la biogenèse des ribosomes, la UsnRNP U4 dans l'épissage des pré-ARNm, le complexe télomérase dans la réplication des télomères, et les mRNP SECIS dans la traduction des sélénoprotéines. Comme c'est le cas pour la majorité des RNP eucaryotes, leur assemblage, sous forme d'entités fonctionnelles, ne constitue pas un processus autonome et requiert l'intervention de facteurs spécialisés. En basant notre étude sur l'assemblage des snoRNP C/D, dans l'organisme modèle Saccharomyces cerevisiae, et en utilisant des approches de biologie moléculaire, de biochimie et de génétique, nous avons entrepris de caractériser ces événements. Nos travaux ont contribué à identifier un ensemble de protéines, agissant de façon coordonnée au sein d'une machinerie conservée entre la levure et l'homme. Cette dernière est composée de deux principales sous-unités : (i) Rsa1p/NUFIP, une protéine plate-forme, qui interagit avec certaines protéines de la famille L7Ae et facilite l'assemblage des RNP, (ii) le complexe R2TP (Rvb1p/TIP49, Rvb2p/TIP48, Pih1p/PIH1, Tah1p/SPAGH), qui pourrait opérer des remodelages conformationnels nécessaires à la formation des RNP matures. En plus de ces acteurs centraux, d'autres facteurs sont apparus intimement liés à ce mécanisme. La protéine Hit1p/TRIP3, interagit notamment avec Rsa1p/NUFIP et s'est avéré requise pour assurer sa stabilité chez la levure. La chaperonne HSP90, dont le rôle est prédominant chez l'homme, exerce son activité sur certains constituants des RNP. Enfin, la protéine Bcd1p/BCD1 pourrait être associée à cette machinerie dans le cadre spécifique de l'assemblage des snoRNP C/DThe L7Ae family proteins are essential components of many RNPs. In vertebrates, C/D and H/ACA snoRNPs are involved in ribosome biogenesis, the U4 snRNP in pre-mRNA splicing, the telomerase complex in telomeres replication, and mRNP SECIS in selenoproteins translation. Like most eukaryotic RNPs, assembly in functional entities is not an autonomous process and requires the intervention of specialized factors. Basing our study on the assembly of C/D snoRNP in the model organism Saccharomyces cerevisiae, and using approaches of molecular biology, biochemistry and genetics, we undertook to decipher these mechanisms. Our work has helped to identify a set of proteins, acting in a coordinated manner within a machinery conserved between yeast and human. This machinery consists of two major subunits: (i) Rsa1p/NUFIP, a platform protein that interacts with some proteins of the L7Ae family and facilitates the RNPs assembly, (ii) the R2TP complex (Rvb1p/TIP49, Rvb2p/TIP48, Pih1p/PIH1, Tah1p/SPAGH), which could induce conformational remodeling necessary for the formation of mature RNPs. In addition to these key players, other factors appeared closely linked to this mechanism. The Hit1p/TRIP3 protein interacts with Rsa1p/NUFIP and is required to ensure its stability in yeast. HSP90 chaperone, whose role is predominant in human, operates on some components of the RNPs. Finally, the Bcd1p/BCD1 protein is associated specifically with this machinery during C/D snoRNPs assembly
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Dodré, Maxime. "Étude du rôle du complexe SMN dans l’assemblage de RNP non codantes ubiquitaires : la SRP, les RNP C/D et H/ACA dont la télomérase, et étude du taux des facteurs d’assemblage de la télomérase dans les cellules cancéreuses." Thesis, Université de Lorraine, 2014. http://www.theses.fr/2014LORR0207.
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Les particules ribonucléoprotéiques (RNP) sont impliquées dans divers mécanismes cellulaires : les UsnRNP, la SRP, les RNP à boîtes C/D et H/ACA dans la modification des ARN et la maturation des ARN ribosomiques, et la télomérase dans le maintien des extrémités chromosomiques. L'assemblage de ces RNP est un processus complexe faisant intervenir de nombreux facteurs, dont le complexe SMN. Un déficit de l’une des protéines de ce complexe conduit à l’amyotrophie spinale. Il est essentiel à la survie cellulaire et est nécessaire à l’assemblage des UsnRNP et de la SRP. Il est suggèré que le complexe SMN joue un rôle dans la biogenèse des RNP à boîtes C/D et H/ACA. Nous avons montré des interactions in vitro et des associations in cellulo entre le complexe SMN et la protéine NUFIP (un facteur d’assemblage de ces RNP). Ces résultats suggèrent l’existence d’un lien fonctionnel entre le complexe SMN et NUFIP dans l'assemblage des RNP à boîtes C/D et H/ACA et de la snRNP U4. Des interactions in vitro entre le complexe SMN et la protéine NAF1 (un facteur d’assemblage des RNP à boîtes H/ACA) ont révélés, que le complexe SMN est capable de s’associer avec la RNP à boîtes H/ACA en formation. Si le complexe SMN intervient dans l’assemblage des RNP, on peut supposer que cet assemblage soit défectueux dans la SMA. Nous avons montré que certains ARN sont accumulés dans la moelle épinière et le cerveau de souris SMA. La télomérase est réactivée dans les cellules cancéreuses. En collaboration avec l’équipe de J-M Vignaud (CHU central, Nancy), nous avons montré une augmentation du taux des protéines cœur des RNP à boîtes H/ACA et de NUFIP dans les cellules tumoralesRibonucleoprotein particles (RNPs) are involved in various cellular mechanisms in eukaryotic cells: UsnRNP, SRP, C/D and H/ACA box RNPs in RNA modifications and rRNA maturation and telomerase in the synthesis of the chromosome extremities. RNP assembly is a very complex process, which involves numerous factors. One of these factors is the SMN complex. Decreased level of one of its components leads to spinal muscular atrophy. It is essential for cell survival and necessary for UsnRNP and SRP assembly. It is suggested that the SMN complex plays a role in C/D and H/ACA RNP biogenesis. We showed in vitro interactions and in cellulo associations between the SMN complex and the protein NUFIP (an assembly factor of these RNP). These results suggest the existence of a functional link between the SMN complex and NUFIP in the assembly of the C/D and H/ACA box RNPs and the U4 snRNP. In vitro interactions between the SMN complex and the protein NAF1 (an assembly factor of the H/ACA boxes RNPs) revealed, that the SMN complex is capable of joining with the H/ACA boxes RNPs in formation. If the SMN complex intervenes in the RNPs assembly, we can suppose that this assembly is defective in the SMA. We showed that any ARN is accumulated in the spinal cord and the brain of SMA mouse. The telomerase is reactivated in cancer cells. In association with the team of J-M Vignaud (CHU central, Nancy), we showed an increase of H/ACA box RNP proteins and NUFIP in these cancer cells
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Hebras, Jade. "Caractérisation moléculaire du petit ARN nucléolaire SNORD115 : un rôle dans la régulation de l'expression et de la fonction du récepteur à la sérotonine 5-HT2C ?" Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30209.
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Le nucléole des mammifères contient des centaines de petits ARN nucléolaires à boîte C/D (SNORD) dont la grande majorité guide une 2'-O-ribose méthylation sur les précurseurs des ARN ribosomiques (pré-ARNr). Certains SNORD facilitent aussi les clivages que subissent le pré-ARNr ou modifient le petit ARN nucléaire U6. Des travaux récents laissent également entrevoir que certains SNORD interagissent avec des ARNm. C'est le cas par exemple pour SNORD115 qui est au cœur de mon travail de thèse. SNORD115 est exprimé uniquement dans le cerveau à partir de nombreux gènes répétés en tandem situés au locus SNURF-SNRPN dont l'expression est contrôlée par l'empreinte génomique parentale. Des défauts génétiques associés à ce locus chromosomique sont associés à une maladie rare: le syndrome de Prader-Willi (SPW). SNORD115 est remarquable car il possède une longue complémentarité conservée avec l'ARNm codant un récepteur à la sérotonine, le variant 5-HT2C. Certains travaux proposent que SNORD115 régule la voie 5-HT2C en modulant l'épissage alternatif ou l'édition A vers I du pré-ARNm 5-HT2C. Un défaut dans l'activité du 5-HT2C pourrait être à l'origine de l'hyperphagie et/ou des anomalies comportementales qui caractérisent le SPW. Mon projet de thèse principal consistait à éprouver cette hypothèse grâce à un nouveau modèle murin CRISPR/Cas9 invalidé pour SNORD115. Mes résultats montrent que la perte d'expression de SNORD115 ne perturbe pas la régulation post-transcriptionnelle du pré-ARNm 5-HT2C in vivo. D'autre part, des études réalisées dans l'équipe n'ont pas permis de révéler des anomalies marquées dans les phénotypes anxio-dépressifs, ni dans le comportement alimentaire. Ma thèse soulève donc des questions importantes quant au rôle régulateur de SNORD115 dans le cerveau et de sa contribution potentielle dans l'étiologie du SPW. En parallèle, j'ai aussi abordé le répertoire des 2'-O-méthylations de l'ARNr dans des tissus murins, notamment le cerveau. Ce travail s'inscrivait dans la thématique émergente de la théorie du "ribosome spécialisé" qui propose qu'une hétérogénéité structurale des composants du ribosome puisse se traduire par des changements dans les capacités fonctionnelles du ribosome. Mes résultats montrent des variations dans la méthylation pour un nombre très limité de sites, et ce principalement au cours du développement. Aussi, les ribosomes des tissus développementaux sont globalement moins méthylés que ceux des tissus adultes. Nous avons concentré nos efforts sur LSU-G4593 dont la méthylation guidée par SNORD78 est retrouvée uniquement au cours du développement. Nous proposons que des évènements d'épissage alternatif du gène-hôte de SNORD78 modulent la production de SNORD78, et de fait le niveau de méthylation LSU-Gm4593. Grâce à l'étude d'une lignée cellulaire humaine (HEK293) invalidée pour SNORD78, j'ai recherché les implications fonctionnelles de LSU-Gm4593. A ce jour, mes travaux ne montrent pas un rôle marqué dans la prolifération cellulaire, ni dans la fidélité de la traduction. La fonction précise de LSU-Gm4593 demeure donc incompriseThe nucleolus of mammalian cells contains hundreds of box C/D small nucleolar RNAs (SNORDs). Majority of them, guide sequence-specific 2'-O ribose methylations into ribosomal RNA (rRNA). Some of them facilitate RNA folding and cleavages of ribosomal RNA precursors or guide ribose methylations into spliceosomal small nuclear RNA U6. Recent studies propose that some SNORD could target other transcripts, possibly messenger RNA as suggested by the brain-specific SNORD115. SNORD115 is processed from tandemly repeated genes embedded in the imprinted SNURF-SNRPN domain. Defects in gene expression at this domain are causally linked to rare disease: the Prader-Willi Syndrome (PWS). Excitingly, SNORD115 displays an extensive region of complementary to a brain-specific mRNA encoding the serotonin receptor 5-HT2C. SNORD115 could influence 5-HT2C signaling by fine-tuning alternative splicing or A to I RNA editing of 5-HT2C pre-mRNA. Reduced 5-HT2C receptor activity could contribute to impaired emotional response and/or compulsive overeating that characterized the syndrome. My work was to test this hypothesis using a CRISPR/Cas9-mediated SNORD115 knockout mouse model. My results show that loss of SNORD115 expression, in vivo, does not alter the post-transcriptional regulation of 5-HT2C pre-mRNA processing. Others results from the team do not reveal any defects in anxio-depressive phenotypes and eating behaviour. Our study questions the regulatory roles of SNORD115 in brain functions and behavioural disturbance associated with PWS. On other hand, I have studied ribose methylation sites in rRNA from mouse tissues. This work was included in emerging field of the specialized ribosome hypothesis which suggests heterogeneity in ribosomes may impact activity of ribosomes. Our results show significant changes at few discrete set of sites, especially in rRNA from developing tissues. Also, rRNA from developing tissues is globally less methylated than rRNA from adult tissues. We focus on LSU-Gm4593 site because this position is specifically methylated only during development and hardly ever detected in adult tissues. Methylation at LSU-G4593 is guided by SNORD78. We propose that the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the host-gene and to correlate with the methylation level of its target site at LSU-G4593. We've used a human cell line (HEK293T) inactivated for the SNORD78 gene in order to understand the functionally role of the corresponding ribose methylation. Our work did not demonstrate any overt cellular phenotypes, even though translation fidelity and the precise function of LSU-Gm4593 remains unknown
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Huttin, Alexandra. "Étude des interactions protéine-protéine entre le complexe de Survie des MotoNeurones (SMN) et les facteurs d'assemblage des RNP à boîtes C/D et H/ACA." Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0250.
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Les particules ribonucléoprotéiques (RNP) à boîtes C/D et H/ACA sont impliquées dans la maturation des UsnRNA et des précurseurs des ARNr. L'assemblage de ces RNP dans les cellules est un processus complexe faisant intervenir de nombreux facteurs cellulaires dont NUFIP, commun aux deux RNP, et NAF1, spécifique aux RNP à boîtes H/ACA. Le complexe de Survie des Motoneurones (SMN) est essentiel à la survie cellulaire et est nécessaire à l'assemblage d'une autre RNP, les UsnRNP, composants des spliceosomes. Un déficit en protéine SMN conduit à une pathologie grave, l'amyotrophie spinale. Plusieurs études suggèrent que le complexe SMN puisse également jouer un rôle dans l'assemblage des RNP à boîtes C/D et H/ACA. Dans le but d'obtenir de plus amples informations, nous avons testé si des interactions existent entre les constituants du complexe SMN et i) les protéines associées aux RNP matures, ainsi que ii) les autres facteurs d'assemblage déjà connus. Ainsi, par une approche de double hybride chez la levure, nous avons observé des interactions fortes entre NAF1 et les protéines Gemin3 et Gemin8 du complexe SMN. Comme la protéine coeur GAR1 des RNP à boîte H/ACA interagit avec la protéine SMN, ces données suggèrent que le complexe SMN participe à l'échange de NAF1 par GAR1, qui est une étape clé de la biogenèse des RNP à boîtes H/ACA. De plus, nous avons mis en évidence des interactions entre Gemin3/NUFIP, Gemin4/NUFIP et Gemin6/NUFIP. L'étude de cette dernière interaction a été approfondie. Nous avons montré que l'interaction est directe, qu'elle existe dans les cellules de mammifères à la fois dans le cytoplasme et le noyau, et nous avons défini les domaines de chaque protéine nécessaires à l'interaction, en collaboration avec l'équipe d'E. Bertrand (IGM Montpellier). Ces résultats ouvrent de larges perspectives quant à un lien fonctionnel entre le complexe SMN et NUFIP dans l'assemblage des RNP à boîtes C/D et H/ACA, mais aussi dans l'assemblage de la snRNP U4 et dans le mécanisme de traduction localisée dans les cellulesBox C/D and H/ACA ribonucleoparticles (RNPs) are required for UsnRNA and ribosomal RNA maturation. Their assembly in cells is a complex process, which implicates numerous cellular factors, such as NUFIP, a common assembly factor, and NAF1, which is a specific factor for H/ACA box RNP assembly. The Survival of Motoneurons (SMN) complex is essential for cell survival and is required for the assembly of another class of RNPs, the UsnRNPs, which are essential components of the splicing machinery. Decreased levels of the SMN protein lead to a severe disease, the spinal muscular atrophy. Several studies led to the proposal that the SMN complex also plays a role in the assembly of box C/D and H/ACA RNPs. In order to obtain more information, we analyzed whether some interactions may exist between components of the SMN complex and i) core proteins of mature RNPs, or ii) factors already known to be involved in the assembly. Using a yeast two-hybrid approach, we observed strong interactions between NAF1 and the SMN complex components, Gemin3 and Gemin8. Since the core H/ACA protein GAR1 interacts with the SMN protein, our data suggest that the SMN complex participates to the exchange of NAF1 by GAR1, which is a crucial step of H/ACA box RNP biogenesis. Furthermore, we discovered strong interactions between Gemin3/NUFIP, Gemin4/NUFIP and Gemin6/NUFIP. Concerning the Gemin6/NUFIP interaction, we showed that is direct, that it exists in both compartments in mammalian cells and we defined domains of both proteins necessary for the interaction in collaboration with the E. Bertrand team (IGM Montpellier). These results open new perspectives concerning functional links between the SMN complex and NUFIP in box H/ACA and C/D RNP assembly, but also in U4 snRNP assembly and in the mechanism of localized translation
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Debieux, Charles Maurice. "The role of SMN in box C/D snoRNP biogenesis." Thesis, University of Newcastle Upon Tyne, 2009. http://hdl.handle.net/10443/274.
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Box C/D small nucleolar ribonucleic protein complexes (snoRNPs) either direct 2’-O-methylation of the pre-ribosomal RNA (pre-rRNA) or function as chaperones in pre-rRNA processing. It has been demonstrated that box C/D snoRNP biogenesis is a highly intricate and co-ordinated process that is mediated by a large, dynamic complex, known as the pre-snoRNP. This complex contains the box C/D snoRNA and a number of common core proteins, which become stably associated with the snoRNA, as well as factors linked to assembly, 3’ RNA processing and transport. Interestingly, the product of the gene linked to the neurodegenerative disorder spinal muscular atrophy, the survival of motor neurons protein (SMN), interacts with the box C/D snoRNP common core protein fibrillarin and is vital for the cellular localisation of both fibrillarin and box C/D snoRNA. The SMN protein operates with Gemins2-8 and UNRIP in what is termed the SMN complex, which has known functions in the assembly of the spliceosomal small nuclear RNPs (snRNPs). As the SMN protein interacts with fibrillarin and is also required for the cellular localisation of both fibrillarin and box C/D snoRNA this study set out to investigate the association of fibrillarin with the box C/D snoRNPs and the role of the SMN complex in this process. In this study it was revealed that the C terminal domain of fibrillarin is essential for cellular localisation and interactions with the box C/D snoRNP assembly factors, which suggests that this domain may mediate fibrillarin association with the box C/D snoRNPs. Also in this study the SMN protein was shown to interact with the box C/D snoRNP assembly factors NUFIP and BCD1, which are stable components of the pre-snoRNP. Analysis of the interaction of SMN with the pre-snoRNP, however, indicates that if SMN does associate with the pre-snoRNP then it is only a transient interaction. Further analysis revealed that as well as the SMN protein, Gemin2, 5, 6 and 7 are essential for the localisation of box C/D snoRNA and that Gemin5 is also required for the accumulation of box C/D snoRNA. This study strengthens the case that the SMN complex is involved in box C/D snoRNP biogenesis, with the data suggesting that it functions as a transport factor rather than in the association of fibrillarin with the box C/D snoRNPs. Analysis of the snRNP transport factor, snurportin1, revealed that it also interacts with numerous components of the pre-snoRNP; however, does not interact with SMN.
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Sun, Jiajun. "In vitro functional and structural studies of archaeal Box C/D RNPS /." Available to subscribers only, 2008. http://proquest.umi.com/pqdweb?did=1650501191&sid=6&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Thesis (M.S.)--Southern Illinois University Carbondale, 2008."Department of Molecular Biology, Microbiology and Biochemisty." Includes bibliographical references (p. 61-66). Also available online.
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Ludwig, Sarah [Verfasser]. "Molekülmechanische Untersuchungen zur Struktur, Funktion und Dynamik der Hepatitis C viralen RNA-abhängigen RNA-Polymerase / Sarah Ludwig." Halle, 2018. http://d-nb.info/1175950548/34.
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Fehr, Carmen [Verfasser]. "Interaktionspartner der Hepatitis C Virus-RNA und Zellzykluskontrolle / Carmen Fehr." Gießen : Universitätsbibliothek, 2011. http://d-nb.info/106311036X/34.
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Yang, Liuqing. "A Novel Function of DEAD Box p68 RNA Helicase In Tumor Cell Proliferation And Epithelial-Mesenchymal Transition." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/biology_diss/8.
Full textAbstract:
Activities of the DEAD box (Asp-Glu-Ala-Asp) family of proteins- including RNA-dependent ATPase and RNA helicase- function in all organisms to sculpt RNA-RNA duplex and RNA-protein complexes, ensuring that necessary rearrangements are rapidly and properly resolved during genetic information processing. Identified as a prototypic member of the DEAD box family and documented as an ATPase and RNA helicase, p68 plays essential and diverse functions in the control of gene expression ranging from pre-mRNA/rRNA processing and mRNA decay/stability to transcriptional activation and initiation. Despite the early implied roles in organ maturation and tumor progression, the functional contributions of p68 to growth/differentiation regulation and cancer development remain undefined. Here, we show c-Abl-dependent phosphorylation of p68 markedly associates with abnormal cell growth and cancer development. Importantly, we characterize an unanticipated signaling module through which p68 functionally contributes to Epithelial-Mesenchymal Transition (EMT) and cell proliferation. p68, which appears to be phosphorylated by c-Abl at tyrosine 593, consequently promotes an EMT through its ability to recruit â-catenin into cell nucleus via a canonic Wnt/â-catenin axis independent way; accordingly, phosphor-p68 (phosphorylated at tyrosine 593 residue) also stimulates tumor cell growth, which requires the ATPase activity of the protein. These findings define a potential mechanism whereby phosphor-p68 recruits â-catenin into cell nucleus in ATP hydrolysis driven fashion and cooperatively regulates transcriptional programs that control an EMT. The dissertation thus demonstrates a tight coordination between DEAD box RNA helicase and cancer development.
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Kharel, Parinati. "Structural And Functional Characterization of Archaeal Fibrillarin (C/D box protein) in Vivo." OpenSIUC, 2013. https://opensiuc.lib.siu.edu/theses/1328.
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Fibrillarin, the methyltransfersase for 2'-O-methylation modification in RNAs is the catalytic component of RNP complexes. Most of the information on the role of fibrillarin in Archaea is based on bioinformatics and in vitro data. The essentiality of this protein was not known in Archaea. Similarly, residues important for 2'-O-methylation in vivo condition were not known in this domain of life. In this study, we demonstrated that fibrillarin is not essential in Haloferax volcanii. Similarly, we showed that Gm1934 modification of large subunit of rRNA is fibrillarin mediated. Using the crystal structure of Pyrococcus furiosus as a template, we created a homology model of Haloferax volcanii fibrillarin . By combining bioinformatics and biochemical approaches, we identified Hv fibrillarin residues important 2'-O-methylation modification in vivo condition.
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Klostermeier, Ulrich C. [Verfasser]. "Tiefencharakterisierung des intestinalen Transkriptoms der Maus mittels RNA-Seq / Ulrich C. Klostermeier." Kiel : Universitätsbibliothek Kiel, 2012. http://d-nb.info/1026442729/34.
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Reich, Stefan [Verfasser], S. E. [Akademischer Betreuer] Behrens, G. [Akademischer Betreuer] Fischer, and K. [Akademischer Betreuer] Tittmann. "Biophysikalische Untersuchungen zur Funktion der Hepatitis C viralen RNA-abhängigen RNA-Polymerase / Stefan Reich. Betreuer: S.-E. Behrens ; G. Fischer ; K. Tittmann." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2012. http://d-nb.info/1029083665/34.
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Schreieck, Amelie [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Role of the RNA polymerase II C-terminal domain in transcription termination and function of Spt5 in 3' RNA-processing factor recruitment / Amelie Schreieck. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1046503308/34.
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Shalamova, Lyudmila [Verfasser]. "Dissecting the requirements for Hepatitis C Virus RNA synthesis using a minus strand replication system / Lyudmila Shalamova." Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1175873535/34.
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Santos, Sara F. C. [Verfasser], Jörg [Gutachter] Vogel, Alexander J. [Gutachter] Westermann, and Jay [Gutachter] Hinton. "Expanding the targetome of Salmonella small RNA PinT using MS2 affinity purification and RNA-Seq (MAPS) / Sara F. C. Santos ; Gutachter: Jörg Vogel, Alexander J. Westermann, Jay Hinton." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1238018351/34.
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Frank, Deborah Jean. "Regulation of cell growth in C. elegans and D. melanogaster by ncl-1/brat /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5029.
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Burke, Paula Louise, and University of Lethbridge Faculty of Arts and Science. "The oligomeric state of archaeal fibrillarin : implications in the organization and function of essential box C/D sRNP particles." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2006, 2006. http://hdl.handle.net/10133/540.
Full textAbstract:
Several vital cellular processes are preformed by large ribonucleoprotein (RNP)
complexes. In archaeal and eukaryotic cells one example of these essential RNP particles
is the box C/D sRNP. In archaea, this complex is responsible for methylation of
ribosomal RNA (rRNA) and transfer RNA (tRNA) during their maturation. Archaeal
fibrillarin (aFib) is the 2'-O methyltransferase responsible for catalysis by this complex.
In this work we have identified the ability of aFib from Sulfolobus acidocaldarius to
form dimers at biologically relevant concentrations and the structural determinants
essential for this association. Based on our model we have predicted the ability of aFibs
to form dimers in different archaeal and eukaryotic species. The ability of aFibs and their
eukaryotic homologs to potentially adopt multiple conformations provides insight into
the dynamics of the box C/D sRNP complex. As observed in the study of other essential
RNP particles, the ability of these complexes to be conformationally diverse is integral to
efficient catalysis of their varied substrates.viii, 74 leaves ; 29 cm.
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Geißler, René [Verfasser], S. E. [Akademischer Betreuer] Behrens, E. [Akademischer Betreuer] Wahle, and M. [Akademischer Betreuer] Gromeier. "Untersuchungen zur Funktion der DEAD-Box-RNA-Helikase DDX3 in der cap- und IRES-abhängigen Translation / René Geißler. Betreuer: S.-E. Behrens ; E. Wahle ; M. Gromeier." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2012. http://d-nb.info/1026043123/34.
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Graziadei, Andrea [Verfasser], and Teresa [Akademischer Betreuer] Carlomagno. "The mechanism and regulation of rRNA methylation by the Box C/D sRNP enzyme in solution / Andrea Graziadei ; Betreuer: Teresa Carlomagno." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1222677679/34.
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Mück, Fabian [Verfasser], Rolf [Gutachter] Marschalek, and Robert [Gutachter] Fürst. "Die DEAD-Box RNA-Helikase DDX6 rekrutiert P-TEFb aus dem 7SK snRNP für die Inkorporation in den AF4-Superelongationskomplex / Fabian Mück. Gutachter: Rolf Marschalek ; Robert Fürst." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2016. http://d-nb.info/1112601422/34.
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Appel, Cathryn Denise. "Structural Features of the Guide:Target RNA Duplex Required for Archaeal C/D sRNA Guided Nucleotide 2?-O-methylation." NCSU, 2006. http://www.lib.ncsu.edu/theses/available/etd-08162006-142559/.
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Archaeal box C/D sRNAs guide the 2?-O-methylation of target nucleotides in both ribosomal and tRNAs. These small non-coding RNAs are characterized by conserved terminal box C/D and internal C?/D? RNA motifs. Each RNA motif binds three core proteins to establish individual RNP complexes that catalyze the site-specific 2?-O-methylation of target nucleotides. Specificity of nucleotide modification is determined by target RNA base pairing with complementary sRNA D or D? guide sequences. The fifth target nucleotide upstream from the D or D? box within the guide:target RNA is then methylated by the core proteins. In vitro assembly of Methanocaldococcus jannaschii sR8 box C/D RNA with recombinant core proteins, L7, Nop56/58, and fibrillarin produces a methylation-competent sRNP complex. This model box C/D sRNP has now been used to determine the structural features of the guide:target RNA duplex that are important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was essential for nucleotide methylation. Mismatched bases within the guide:target RNA duplex also disrupted target nucleotide methylation. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA helix as target deoxy-oligonucleotides possessing a target ribonucleotide were not methylated. Methylation specificity at the base paired guide:target nucleotide was compromised by elevated Mg2+ concentrations. In high divalent cation concentrations, target nucleotides not hydrogen bonded to the guide nucleotide were nevertheless methylated. Interestingly, D and D? target RNAs were methylated to different levels when deoxynucleotides were inserted within the target RNA or when target methylation was carried out in elevated Mg2+ concentrations. These results suggested that structural features unique to the box C/D and C?/D? RNPs affect their nucleotide methylation capabilities. Finally, the ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested an intrinsic ability of this archaeal RNA:protein enzyme to unwind double-stranded target RNAs prior to nucleotide modification.
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Prieto, Marcela Bach. "Caracterização funcional das proteínas Nop17p e Rsa1p de Saccharomyces cerevisiae." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22012015-145300/.
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Nop17p e Rsa1p são proteínas nucleolares em Saccharomyces cerevisiae, as quais foram identificadas pela sua associação a dois complexos celulares: os snoRNPs de box C/D, através de interação com as subunidades Nop58p e Snu13p, respectivamente, e o R2TP/Hsp90p. Nop17p parece ser responsável por direcionar a chaperona Hsp90p durante a montagem dos snoRNPs, e a associação de Rsa1p a estes complexos ainda não tem uma função estabelecida. Neste trabalho, nós mostramos que a ausência de ambas as proteínas afetam a estabilidade da proteína Nop58p dos snoRNPs e afetam a localização do snoRNA U3. Em relação à ordem de interação das proteínas do core de snoRNps de box C/D, Nop17p associa-se de maneira transiente a Nop1p/Snu13p, seguida da ligação de Nop58p ao complexo. Quanto à rede de interação do R2TP, obtivemos o mutante Nop17(N307S), que não mais interage com Tah1p. Este mutante interage com a subunidade Rvb1p do R2TP, mas não se associa com outras proteínas parceiras de Nop17p(WT). Apesar da importância da interação Nop17p-Tah1p, sua interrupção não afeta o crescimento celular, o que sugere a possibilidade de outro fator estar envolvido na associação entre Nop17p e Hsp90p.Nop17p and Rsa1p are Saccharomyces cerevisiae nucleolar proteins, which were identified for its association with two cellular complexes: box C/D snoRNPs, through interaction with the core subunits Nop58p and Snu13p respectively, and the R2TP/Hsp90p. Nop17p seems to be responsible for directing Hsp90p to the assembly of snoRNPs. The Rsa1p association to these complexes still have no defined function. In this work, we showed that both proteins absence affect Nop58p stability and causes a mislocalization of the U3 snoRNA. Relativel to the order of assembly of the box C/D snoRNPs core proteins, Nop17p associates transiently with Nop1p/Snu13p, followed by the Nop58p joining to the complex. To study in more detail the protein interactions within the R2TP complex, we obtained the Nop17(N307S) mutant, which no longer interacts withTah1p, but still interacts withRvb1p, another R2TP subunit. Nop17(N307S) does not interact with other Nop17p(WT) partners. Despite the importance of the Nop17p-Tah1p association, the disruption of this interaction does not affect cell growth, suggesting the involvement of a second factor on the Nop17p and Hsp90p association.
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Kisielnicka, Edyta. "SCF-mediated degradation of the two translational regulators, CPB-3 and GLD-1, during oogenesis in C. elegans." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-234186.
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The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-‐transcriptional level. This relies on RNA-‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-‐binding proteins abundance in the context of germ cell development.
This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-‐binding proteins, CPB-‐3 and GLD-‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-‐3/CPEB and GLD-‐1/STAR at the pachytene-‐to-‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-‐3 and GLD-‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-‐activated protein kinase, MPK-‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-‐1 signaling may couple RBP-‐mediated regulation of gene expression to progression through meiosis during oogenesis.
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Winkler, Eileen-Christin [Verfasser]. "Einfluss der RNA-Replikation des Hepatitis-C-Virus auf die Expression des vascular endothelial growth factor A / Eileen-Christin Winkler." Halle, 2017. http://d-nb.info/1150704470/34.
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Nist, Richard Neil. "Maturation of tRNA in Haloferax volcanii." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1308066223.
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Schumann, Nadine [Verfasser], D. [Akademischer Betreuer] Scheel, H. B. [Akademischer Betreuer] Deising, and S. A. [Akademischer Betreuer] Rensing. "Phylogenetische und molekulare Charakterisierung pflanzlicher F-Box-Proteine mit C-terminaler Kelch-Repeat-Domäne / Nadine Schumann. Betreuer: D. Scheel ; H. B. Deising ; S. A. Rensing." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2013. http://d-nb.info/1043480560/34.
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Paieri, Francesca [Verfasser], and Dario [Akademischer Betreuer] Leister. "Expression of the plant Photosystem II core proteins in the cyanobacterium Synechocystis sp. PCC6803 and characterization of the DEAD-box RNA helicase RH50 of A. thaliana / Francesca Paieri ; Betreuer: Dario Leister." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1192215362/34.
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Lapuschkin, Sebastian Verfasser], Klaus-Robert [Akademischer Betreuer] [Gutachter] [Müller, Thomas [Gutachter] Wiegand, and Jose C. [Gutachter] Principe. "Opening the machine learning black box with Layer-wise Relevance Propagation / Sebastian Lapuschkin ; Gutachter: Klaus-Robert Müller, Thomas Wiegand, Jose C. Principe ; Betreuer: Klaus-Robert Müller." Berlin : Technische Universität Berlin, 2019. http://d-nb.info/1177139251/34.
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Lapuschkin, Sebastian [Verfasser], Klaus-Robert [Akademischer Betreuer] [Gutachter] Müller, Thomas [Gutachter] Wiegand, and Jose C. [Gutachter] Principe. "Opening the machine learning black box with Layer-wise Relevance Propagation / Sebastian Lapuschkin ; Gutachter: Klaus-Robert Müller, Thomas Wiegand, Jose C. Principe ; Betreuer: Klaus-Robert Müller." Berlin : Technische Universität Berlin, 2019. http://d-nb.info/1177139251/34.
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Roth, Bernhard [Verfasser], and Ralf C. [Gutachter] Bargou. "Entwicklung von Plasmiden mit multiplen short hairpin RNA-Expressionskassetten für den simultanen Knockdown onkogener Zielstrukturen im Multiplen Myelom / Bernhard Roth ; Gutachter: Ralf C. Bargou." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1225295939/34.
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Sherrard, Ryan W. [Verfasser], and Barbara [Akademischer Betreuer] Conradt. "Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development / Ryan W. Sherrard ; Betreuer: Barbara Conradt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1180981820/34.
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Sherrard, Ryan William [Verfasser], and Barbara [Akademischer Betreuer] Conradt. "Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development / Ryan W. Sherrard ; Betreuer: Barbara Conradt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1180981820/34.
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Nieder-Röhrmann, Anika [Verfasser]. "Separate Betrachtung der beiden miR-122-Bindungsstellen in der Hepatitis C Virus 5´-UTR im Hinblick auf die Translationsstimulation und RNA-Stabilität / Anika Nieder-Röhrmann." Gießen : Universitätsbibliothek, 2016. http://d-nb.info/111900960X/34.
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Dünnes, Nadia [Verfasser]. "Analyse der Interaktion von microRNA-122-Protein-Komplexen mit der NS5B-kodierenden Region und der 3´-untranslatierten Region der Hepatitis C Virus-RNA / Nadia Dünnes." Gießen : Universitätsbibliothek, 2016. http://d-nb.info/1118289773/34.
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Husnik, Stefanie [Verfasser], and Thomas von [Akademischer Betreuer] Hahn. "Identifikation von mTORC1 als neuen Wirtsfaktor für die Hepatitis C Virus RNA Replikation / Stefanie Husnik ; Akademischer Betreuer: Thomas von Hahn ; Klinik für Gastroenterologie, Hepatologie und Endokrinologie." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2020. http://d-nb.info/1207772801/34.
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Neumann, Bettina [Verfasser], Heike [Akademischer Betreuer] Krebber, and Gerhard [Akademischer Betreuer] Braus. "Studies on the DEAD-box RNA-helicase Dbp5 and the ABC-protein Rli1 in translation termination and identification of a novel function of Dbp5 in ribosomal transport / Bettina Neumann. Gutachter: Heike Krebber ; Gerhard Braus. Betreuer: Heike Krebber." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1076161030/34.
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Tiotiu, Decebal. "Analyse fonctionnelle des protéines Hit1 et Bcd1 impliquées dans la biogenèse des snoRNP à boîtes C/D eucaryotes." Thesis, Université de Lorraine, 2016. http://www.theses.fr/2016LORR0319/document.
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Chez les eucaryotes, la biogenèse des ribosomes débute dans le nucléole par la maturation et la modification des ARN ribosomiques (ARNr), et fait intervenir des centaines de particules ribonucléoprotéiques (RNP) distinctes, comme les petites RNP nucléolaires (snoRNP) à boîtes C/D, qui portent une activité méthyl transférase ciblée sur la position 2’-OH des riboses. Leur biogenèse nécessite l’intervention transitoire de facteurs protéiques constituant une machinerie d’assemblage spécifique. Mon travail de thèse a visé à étudier le rôle fonctionnel de deux de ces facteurs chez la levure S. cerevisiae les protéines Hit1 et Bcd1. Hit1p avait été trouvée au laboratoire être impliquée dans la biogenèse des snoRNP à boîtes C/D, et il était connu que l’expression de Bcd1p est essentielle à la viabilité cellulaire et pour la stabilité des snoRNA à boîtes C/D. Lors de ce travail, nous avons retrouvé le domaine fonctionnel de Hit1p et identifié les acides aminés impliqués dans l’interaction avec Rsa1p, un autre facteur d’assemblage. Par une approche similaire, nous avons recherché les domaines nécessaires à la fonctionnalité de Bcd1p. Le mécanisme par lequel Bcd1p influence spécifiquement les taux de snoRNA à boîtes C/D reste inconnu, mais au cours de ce travail j’ai identifié un nouveau partenaire potentiel pour cette protéine - la chaperonne d’histone Rtt106p. La dernière partie de mon travail a visé à rechercher le lien fonctionnel entre Rtt106p et l’expression des snoRNA à boîtes C/DIn eukaryotes, ribosome biogenesis begins in the nucleolus, by maturation and modification of ribosomal RNAs (rRNA) and involves hundreds of distinct ribonucleoprotein particles, like box C/D small nucleolar RNPs (snoRNPs). Their assembly requires the transient intervention of protein factors constituting a specific assembly machinery. My PhD work aimed to investigate the functional role of two such factors, Bcd1p and Hit1p, in the yeast S. cerevisiae. Hit1p involvement in box C/D snoRNP biogenesis was revealed in our lab, and it was known that Bcd1p expression is essential to cell viability and box C/D snoRNA stability. During this work, we identified the functional domain of Hit1p, and the aminoacids involved in its interaction with Rsa1, another assembly factor. By a similar approach we identified the functional domains of Bcd1p. The mechanism by which Bcd1p specifically influences box C/D snoRNA levels is unknown. However, I identified a potentially new partner for this protein – the Rtt106p histone chaperone. The last part of my work aimed to search for a functional link between this histone chaperone and box C/D snoRNA expression
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Bizarro, Jonathan. "Rôle des facteurs d'assemblage et du système HSP90/R2TP dans la biogenèse des particules C/D snoRNP et U4 snRNP." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20075.
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La machinerie d'assemblage HSP90/R2TP est impliquée dans la biogenèse de complexes essentiels à l'expression génique et à la croissance cellulaire. Le complexe est constitué des protéines PIH1D1, RPAP3 et des AAA+ ATPases RVB1 et RVB2. Le R2TP, via son adaptateur NUFIP, permet l'assemblage de complexes ribonucléoprotéiques tels que les snoRNP à boîtes C/D, et le snRNP U4, tous deux impliqués respectivement dans la maturation des ARNr et ARNm. Le mode d'action du R2TP dans ces processus n'était pas bien compris. Pour étudier cela, une approche de protéomique, avec des tests d'interaction ARN/protéine et protéine/protéine, ainsi qu'une approche structurale, ont été utilisés. Un nouveau modèle a ainsi été établi. Le R2TP permettrait de former des pré-complexes d'assemblage contenant les protéines cœurs du complexe RNP avec des facteurs d'assemblage mais sans l'ARN. Les protéines RVB se détacheraient du R2TP pour rester associées à ce pré-complexe d'assemblage, par la suite, elles permettraient de le stabiliser tout en y incorporant de nouvelles protéines cœurs, et en relargant des facteurs d'assemblage ayant déjà accompli leur fonction dans le processus de biogenèse. Cette fonction de chaperon moléculaire des complexes en cours d'assemblage est très probablement régulée par hydrolyse de l'ATP par les ATPases RVB, et ceci sous le contrôle de co-facteurs, comme potentiellement la protéine BCD1. Dans le cas de l'assemblage des C/D snoRNP, il a été établi un modèle d'assemblage dans lequel le rôle des différents facteurs d'assemblage peut être prédit. ZNHIT3 aurait un rôle dans l'incorporation du snoARN naissant dans le pré-complexe, et NUFIP permettrait de garder la particule immature dans une conformation inactive afin de faciliter l'obtention de la structure active du snoRNP grâce aux RVB. Avec les travaux sur la biogenèse de la particule U4, il a été mis en évidence l'existence d'un pré-complexe cytoplasmique contenant PRP31/NUFIP/R2TP/complexe SMN qui serait important pour l'assemblage de la snRNP U4 avec non seulement les protéines Sm, mais aussi la protéine PRP31The HSP90/R2TP machinery is involved in the biogenesis of essential complex for gene expression and cell growth. The complex consists of proteins PIH1D1, RPAP3 and the AAA+ ATPases RVB1 and RVB2. The R2TP, via its NUFIP adaptator, allows assembly of ribonucleoprotein complexes like box C/D snoRNP, and the U4 snRNP, both involved in the maturation of mRNA and rRNA respectively. The mode of action of R2TP in these processes is not well understood. In this study, a proteomic approach, with tests of interaction RNA/protein and protein/protein and a structural approach, were used. A new model has been established. The R2TP would form an assembly pre-complex containing RNP core proteins with assembly factors but not RNA. RVB proteins detach from R2TP to remain associated with the assembly pre-complex, and then, would stabilize it while incorporating new core proteins. They would also release assembly factors that already have accomplished their function in the biogenesis process. This function of molecular chaperone complex during assembly is most likely regulated by ATP hydrolysis by the RVB ATPases, and this under the control of co-factors as potentially BCD1 protein. In the case of the assembly of box C/D snoRNP, it was established an assembly model in which the roles of the various assembly factors can be predicted. ZNHIT3 has a role in the incorporation of the nascent snoRNA in the pre-complex and NUFIP would keep the immature particle into an inactive conformation to facilitate the formation of the active structure of the snoRNP through RVB. With the study of the biogenesis of the U4 particle, it was revealed the existence of a cytoplasmic pre-complex containing PRP31/NUFIP/R2TP/SMN complex that would be important for the assembly of the U4 snRNP with not only Sm protein but also the PRP31 protein
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Penzel, Anika [Verfasser], Sven-Erik [Akademischer Betreuer] Behrens, Stefan [Akademischer Betreuer] Hüttelmaier, and Gregor [Akademischer Betreuer] Meyers. "Einfluss der RNA-Replikation des Hepatitis-C-Virus auf die Degradation der zellulären mRNA des vascular endothelial growth factor-A / Anika Penzel. Betreuer: Sven-Erik Behrens ; Stefan Hüttelmaier ; Gregor Meyers." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2015. http://d-nb.info/1078017417/34.
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Alhagdow, Moftah Mohamed. "Caractérisation fonctionnelle de la GDP-D-mannose-3,5-épimérase et galactono-1,4-lactone déshydrogénase, enzymes de la voie de biosynthèse de la vitamine C chez la tomate." Bordeaux 1, 2006. http://www.theses.fr/2006BOR13162.
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Afin d'étudier l'effet d'une réduction de la teneur en vitamine C sur le développement des plantes, une approche de transgénèse visant à supprimer l'expression de la GDP-mannose-3,5-épimérase (GME) et la galactone-1,4-lactone déshydrogénse (GalLDH), deux enzymes de la biosynthèse, à été réalisée chez la tomate. La caractérisation phénotypique des transformants GalLDH et GME révèle des différences importantes au niveau du développement de la plante du du fruit. L'analyse du transcriptome et du métabolome montre que plusieurs voies de métabolisme primaire et secondaire sont sévèrement affectées. Nos résultats démontrent que, en plus de son rôle antioxydant majeur dans la plante, l'ascorbate peut jouer un rôle important dans la régulation des processus de prolifération et d'expansion cellulaire dans la plante et le fruit.
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Paul, Arnaud. "Rôle de la protéine Bcd1p/BCD1 dans les étapes précoces de la biogenèse des snoRNP à boîtes C/D eucaryotes." Thesis, Université de Lorraine, 2018. http://www.theses.fr/2018LORR0148.
Full textAbstract:
La biogenèse des ribosomes matures et fonctionnels est notamment dépendante de petites particules ribonucléoprotéiques composées d’ARN et de protéines, les snoRNP (small nucleolar RiboNucleoProteins). Celles-ci sont subdivisées en deux familles : les snoRNP à boîtes C/D et les snoRNP à boîtes H/ACA. Ces deux classes de snoRNP catalysent des modifications chimiques, respectivement de 2’-O-méthylation et de pseudouridylation, sur des positions spécifiques des ARN ribosomiques (ARNr), ou sont impliquées dans des clivages du long ARNr précurseur. Les snoRNP à boîtes C/D sont composées d’un snoARN à boîtes C/D servant de guide pour cibler la position à modifier, et d’un jeu invariant de quatre protéines : Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 et Nop58p/NOP58 (levure/Homme). Ces snoRNP sont produites par la cellule grâce à la présence de plusieurs complexes protéiques constituant une machinerie pour leur assemblage. Outre plusieurs facteurs protéiques déjà connus dans la biogenèse de snoRNP à boîtes C/D comme les protéines Rsa1p/NUFIP, Hit1p/ZNHIT3 et les protéines du complexe R2TP, d’autres protéines pourraient compléter cette machinerie. Parmi ces facteurs additionnels, la protéine Bcd1p/ZNHIT6, pour Box C/D snoRNA protein 1, est essentielle pour maintenir spécifiquement la stabilité in vivo des snoARN à boîtes C/D, et des associations ont pu être identifiées entre Bcd1p/ZNHIT6 avec différents partenaires protéiques de la machinerie d’assemblage de ces particules. Toutefois, l’étape d’assemblage où Bcd1p/ZNHIT6 intervient et la fonction qu’elle y accomplit demeurent inconnues. L’utilisation d’outils in vivo et in vitro chez la levure S. cerevisiae et chez les mammifères nous ont permis de progresser dans la compréhension de la fonction de Bcd1p/ZNHIT6 dans l’assemblage des snoRNP à boîtes C/D. Bcd1p est un facteur d’assemblage recruté de manière co-transcriptionnelle sur les loci codant les snoARN à boîtes C/D et est requis pour le recrutement des complexes d’assemblage sur les snoARN en cours de transcription. Plus spécifiquement, Bcd1p affecte l’interaction de Nop58p avec le facteur d’assemblage Rsa1p, suggérant une fonction dans le recrutement de Nop58p dans une pré-snoRNP en cours d’assemblage. Ce travail a permis d’apporter des informations importantes permettant d’expliquer le caractère essentiel de Bcd1p dans la fonction et la biogenèse des snoRNP à boîtes C/DRibosome biogenesis is especially dependent on the action of small RNA/proteins complexes called small nucleolar ribonucleoproteins (snoRNPs). They are divided into two main families: the so-called box C/D snoRNPs and box H/ACA snoRNPs. Each category performs specific enzymatic processes, 2’-O-methylation and pseudouridylation, respectively, and induces target-specific chemical modification on rRNAs. Few snoRNPs are also essential for pre-rRNA processing. The box C/D snoRNPs are formed by the association of a box C/D snoRNA with a set of four invariant proteins: Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 and Nop58p/NOP58 (yeast/Human). Biogenesis of these RNPs relies on the action of several proteins complexes which constitute a dedicated assembly machinery. Rsa1p/NUFIP, Hit1p/ZNHIT3, and components of the R2TP complex are the best characterized protein actors of this machinery. Additional protein factors probably participate in box C/D snoRNP biogenesis; Bcd1p/ZNHIT6 (Box C/D snoRNA protein 1) is such a candidate as it is essential for the in vivo stability of box C/D snoRNAs, and it was found associated with proteins involved in this machinery in yeast and Human. However, the mechanism governing the recruitment of this protein towards the biogenesis of box C/D snoRNP, and the step of the assembly process relying on the presence of Bcd1p are still unknown. In S. cerevisiae and Human, in vivo and in vitro tools allowed us to improve the understanding of the functions of Bcd1p/ZNHIT6 in box C/D snoRNP assembly. Bcd1p is an assembly factor that is recruited co-transcriptionally on box C/D snoRNA loci, and is required for the recruitment of assembly complexes on nascent snoRNAs. Bcd1p is important for Nop58p association with the assembly factor Rsa1p, which suggests that its primary function is to recruit Nop58p to nascent pre-snoRNPs. This work evidenced important information on the essential role of Bcd1p in C/D snoRNP biogenesis and function
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Bragantini, Benoît. "Caractérisation structurale et fonctionnelle de la protéine Bcd1, impliquée dans la biogenèse des snoRNP à boîtes C/D chez la levure Saccharomyces cerevisiae." Thesis, Université de Lorraine, 2016. http://www.theses.fr/2016LORR0295/document.
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La protéine Bcd1 est un facteur nucléaire essentiel à la viabilité cellulaire de la levure Saccharomyces cerevisiae. Il est décrit comme requis pour assurer la stabilité des snoRNA à boîtes C/D. Ces petits ARN non codants s’assemblent à un jeu de 4 protéines invariables pour former les snoRNP à boîtes C/D qui sont des acteurs cruciaux de la biogenèse des ribosomes. En effet, quelques-unes de ces particules participent aux mécanismes assurant la maturation du précurseur des ARN ribosomiques et la grande majorité des autres particules sont des catalyseurs de la modification par 2’-O-méthylation des riboses. Bcd1p n’est pas présente au sein des particules matures, mais fait partie de ses facteurs d’assemblage, au même titre que les sous-complexes Rsa1p:Hit1p et R2TP (Rvb1p:Rvb2p:Tah1p:Pih1p). Notre analyse de différents fragments de Bcd1p a dans un premier temps montré que sa région N-terminale (résidus 1 à 96) suffit à lui conférer son caractère essentiel. Cette région comprend un domaine à double doigt à zinc de la famille zf-HIT, également présent chez un autre facteur d’assemblage des snoRNP à boîtes C/D, la protéine Hit1. Nous avons résolu la structure 3D en solution de ces doigts à zinc et montré que ce sont des modules d’interaction avec les protéines Rvb1/2. Dans un second temps nous avons identifié la région C-terminale (résidus 120 à 303) de la protéine Bcd1 comme étant suffisante pour interagir avec la chaperonne d’histone Rtt106p. La structure 3D en solution de ce domaine a été déterminée par RMN. Différentes approches de cinétique d’échange hydrogène/deutérium et d’expériences de cross-link suivies par des analyses par spectrométrie de masse, des expériences de titrage par RMN et de SAXS nous ont permis d’obtenir des informations sur les surfaces d’interaction de chacune de ces deux protéines. Un fragment, défini à partir des données de RMN de Bcd1p libre, nous a permis d'obtenir des cristaux du complexe Bcd1p:Rtt106p ouvrant la perspective de résoudre sa structure 3D par diffraction aux rayons X. De plus, des études fonctionnelles ont débuté visant à déterminer l’importance de la formation de ce complexe sur la biogenèse des snoRNP à boîtes C/D et l’impact de Bcd1p sur l’interaction entre Rtt106p et les nucléosomesThe protein Bcd1 is a nuclear factor essential for the cellular viability of the yeast Saccharomyces cerevisiae. It is described as required to ensure box C/D snoRNA stability. These small non-coding RNAs associate with an invariable set of 4 proteins to form the box C/D snoRNPs that are crucial players in ribosome biogenesis. Indeed, some of these particles participate in mechanisms for the maturation of the ribosomal RNA precursor (prerRNA) and the vast majority of the other particles are catalysts of 2’-O-methylation of riboses. Bcd1p is not present in mature particles, but is one of the assembly factors in addition to the Rsa1p:Hit1p and R2TP (Rvb1p:Rvb2p:Tah1p:Pih1p) sub-complexes. Our analysis of the different Bcd1p fragments has firstly shown that the essential function of Bcd1p relies on its N-terminal region (residues 1 to 96). It comprises a double zinc finger domain from the zf-HIT family, also present in another box C/D snoRNP assembly factor, the protein Hit1. We solved the 3D solution structure of these two zinc fingers and showed that these are modules for the interaction of Bcd1p with the Rvb1/2 proteins. Secondly, we identified the C-terminal region (residues 120 to 303) of Bcd1p as being sufficient to interact with the histone chaperone Rtt106p. The 3D solution structure of this domain of Bcd1p was determined by NMR. Different approaches of hydrogen/deuterium kinetic exchange and cross-link experiments followed by mass spectrometry analysis, NMR titration, and SAXS allowed us to obtain information about the interaction surfaces on each of the two proteins. A fragment defined from NMR data on the free Bcd1p allowed us to obtain crystals of the Bcd1p:Rtt106p complex, opening the perspective to solve its 3D structure by X-ray diffraction. Furthermore, functional studies started in order to determine the importance of this complex formation in box C/D snoRNP biogenesis and the impact of Bcd1p on the interaction of Rtt106p with nucleosomes