Dissertations / Theses on the topic 'RNA 3' Polyadenylation Signals'

Cleavage and polyadenylation of a precursor mRNA is important for transcription termination, mRNA stability, and regulation of gene expression. This process is directed by a multitude of protein factors and cis elements in the pre-mRNA sequence surrounding the cleavage and polyadenylation site. Importantly, the location of the cleavage and polyadenylation site helps define the 3’ untranslated region of a transcript, which is important for regulation by microRNAs and RNA binding proteins. Additionally, these sites have generally been poorly annotated. To identify 3’ ends, many techniques utilize an oligo-dT primer to construct deep sequencing libraries. However, this approach can lead to identification of artifactual polyadenylation sites due to internal priming in homopolymeric stretches of adenines. Previously, simple heuristic filters relying on the number of adenines in the genomic sequence downstream of a putative polyadenylation site have been used to remove these sites of internal priming. However, these simple filters may not remove all sites of internal priming and may also exclude true polyadenylation sites. Therefore, I developed a naïve Bayes classifier to identify putative sites from oligo-dT primed 3’ end deep sequencing as true or false/internally primed. Notably, this algorithm uses a combination of sequence elements to distinguish between true and false sites. Finally, the resulting algorithm is highly accurate in multiple model systems and facilitates identification of novel polyadenylation sites.

Thesis (Ph. D.)--University of Kentucky, 2005.
Title from document title page (viewed on November 7, 2005). Document formatted into pages; contains vi, 135 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 113-133).

Cytoplasmic polyadenylation is a widespread mechanism to control mRNA translation. In vertebrates, this mechanism requires two sequence elements in the 3’ UTR of substrate mRNAs, the U-rich Cytoplasmic Polyadenylation Element (CPE) and the AAUAAA polyadenylation hexanucleotide (HN). In Drosophila early embryos, the cytoplasmic polyadenylation of Toll mRNA occurs independently of these elements, and requires Dicer-2, a factor previously known for its functions in RNA interference (RNAi), in addition to the poly(A) polymerase Wispy. To understand this novel function of Dicer-2 in cytoplasmic polyadenylation and translation, we aimed to dissect the required cisacting requirements in Toll mRNA, and to identify Dicer-2 protein partners and mRNA targets. We found that multiple signals in the 3’ UTR of Toll cooperate for polyadenylation, and that in early embryos the non-canonical signals dominate, probably due to the presence of inhibitory elements for canonical polyadenylation. Interactome analysis using affinity purification of Dicer-2 and mass spectrometry revealed that Dicer-2 interacts with multiple proteins outside the RNAi pathway. Importantly, proteins involved in poly(A) tailmediated translational regulation and PABP binding were identified, suggesting potential co-factors of Dicer-2 in polyadenylation. Furthermore, RIP-Seq analysis of Dicer-2 revealed enrichment of mRNAs which were previously found downregulated in wispy mutants. These results suggest that the role of Dicer-2 in cytoplasmic viii polyadenylation might be widespread, and provide the basis for future investigation.
La poliadenilación citoplasmática es un mecanismo de control de la traducción extendido a lo largo de la escala animal. En vertebrados, este mecanismo requiere dos secuencias en el extremo 3’ no traducido (UTR) del mRNA, el elemento de poliadenilación citoplasmática rico en uridinas (CPE) y el hexanucleótido de poliadenilación AAUAAA (HN). En embriones tempranos de Drosophila, la poliadenilación de Toll, sin embargo, ocurre independientemente de estas secuencias, y requiere al menos dos factores: Dicer-2, una proteína previamente implicada en interferencia de RNA (RNAi), y la poly(A) polimerasa Wispy. Para entender esta nueva función de Dicer-2 en poliadenilación citoplasmática y traducción, nos propusimos diseccionar los elementos en el extremo 3’ UTR de Toll relevantes para este mecanismo de poliadenilación no canónica, así como identificar proteínas y mRNAs que interaccionan con Dicer-2 a gran escala. Nuestros resultados indican que el extremo 3’ UTR de Toll contiene varias secuencias implicadas en poliadenilación, y que el mecanismo no canónico es dominante en embriones tempranos, probablemente porque los elementos canónicos (CPE y HN) se encuentran activamente reprimidos. El estudio del interactoma de Dicer-2, realizado por purificación por afinidad e identificación por espectrometría de masas, reveló numerosas proteínas que no están implicadas en RNAi. Entre ellas, encontramos factores previamente relacionados con control de la longitud del poly(A) o con interacción con PABP y traducción, sugiriendo que estos factores podrían actuar x como co-factores de Dicer-2 en poliadenilación citoplasmática. Análisis de mRNA targets mediante RIP-Seq reveló multiples tránscritos que previamente fueron identificados como targets de Wispy. Estos resultados sugieren una función extendida de Dicer-2 en poliadenilación citoplasmática, y establecen una base sólida para investigaciones futuras.

Mestrado em Biologia Molecular e Celular
Alternative polyadenylation (APA) is an important mechanism of gene regulation that occurs in 70% of eukaryotic organisms. This process comprises the formation of alternative 3’ ends of an mRNA by cleavage of the pre-mRNA and polyadenylation at different sites according to the polyadenylation signals (pAs). The choice of pAs in APA is a co-transcriptional mechanism that depends on auxiliary cis- and trans-acting factors. The usage of the proximal or the distal pAs has been related to global physiologic events. It is consensually assumed that in proliferative conditions there is preferential usage of proximal pAs, while during development and in differentiated cellular states occurs lengthening of the 3’UTRs by selection of the distal pAs. This pattern is also confirmed in brain tissues, where most of the cells are differentiated, and where it was observed a lengthening of the 3’ UTRs. However, there is not a complete switch for the distal pA, since the shortest mRNA is still expressed. Rho GTPases are key molecular switchers essential for several cellular processes, including differentiation, however nothing is known about transcriptional regulation in these genes. Therefore, we started to explore if Rho GTPases genes undergo APA. We found by 3’RACE analyses, that classical Rho GTPAses express two alternative mRNA isoforms. However during oligodendrocytes differentiation, they preferentially express the shortest mRNA isoform, and we did not observe a switch towards the distal pA usage, in contrast with the published genome-wide data obtained from brain tissues. Since Rho GTPases are tightly regulated at the protein level by GEFs and GAPs, they may not require this mode of co-transcriptional regulation. The atypical RhoBTB2, which is constitutively active, present a global induction of distal pA sites, distinct from the classical Rho GTPases. Interestingly, this pattern suggests that APA is a gene specific mechanism. As longer 3'UTRs contain more binding sites for miRNAs and RNA binding proteins (RBPs) this suggests that atypical Rho GTPases require a fine-tune regulation at the co-transcriptional level, by APA. Additionally, we showed that APA is also cell-specific, by analyzing the expression of the different mRNA isoforms of Rho GTPases in other glial cells (microglia, astrocytes) and different types of neurons (cortical, striatal and hippocampal). We observed the same APA profile for the selected Rho GTPases in all glial cells types. However, in cortical and striatal neurons we observed a lengthening in the 3’UTR Rac1 mRNA during axonal growth, which results in the increase of the total protein levels. Taken together, our results indicate for the first time that APA is a gene- and cell- specific mechanism. In addition, we have found a differential expression of both Cdc42 isoforms during OL and sciatic nerve differentiation. During in vitro OL and in vivo sciatic nerve differentiation we observed an increase in the expression ratio between Cdc42 Iso1/Cdc42 Iso2. Further, constitutive expression of Cdc42 Iso2 in OLs induces a delay in differentiation, whereas constitutive expression of Cdc42 Iso1 induces an increase in OL branching, suggesting an exacerbation of the differentiated phenotype. Thus, these observations suggest a distinct role for the different Cdc42 isoforms during OL differentiation. Overall, this thesis opens new avenues to explore in the future that can impact our understanding on the regulation of the myelination/remyelination processes.
A poliadenilação alternativa (APA) é um mecanismo importante de regulação genética que ocorre em 70% dos organismos eucariotas. Este mecanismo compreende a formação de extremidades 3’ alternativas por poliadenilação em diferentes locais do mRNA, de acordo com os sinais de poliadenilação (pAs). Na APA, a escolha dos pAs é um mecanismo co-transcripcional que depende de factores auxiliares cis e trans necessários para os processos de clivagem e poliadenilação de todos os pré-mRNAs. Além disso, o uso dos pAs proximais ou distais está relacionado com eventos fisiológicos gerais. Consensualmente assume-se que em estados de proliferação ocorre o encurtamento, enquanto em estados de desenvolvimento e diferenciação ocorre o alongamento das extremidades 3’ não traduzidas (3’UTRs). Este padrão de APA é confirmado em tecidos cerebrais, onde a maior parte das células são diferenciadas, no entanto não existe uma alteração completa para a isoforma de mRNA longa uma vez que a isoforma curta continua a ser expressa. As Rho GTPases são ‘interruptores’ moleculares essenciais a vários processos celulares, incluindo a diferenciação, no entanto nada é conhecido sobre a sua regulação transcripcional. Assim, começamos a explorar se estes genes são regulados por APA. Descobrimos por análise de 3´RACE que, as Rho GTPases clássicas, expressam duas formas alternativas de mRNA. Contudo durante a diferenciação dos oligodendrócitos (OLs), eles expressam preferencialmente a isoforma mRNA mais curta, e não se observou uma alteração para a escolha da isoforma mais longa, em contraste com os dados de estudos globais do genoma em tecido cerebral. Uma vez que estas proteínas são altamente reguladas por GEFs e por GAPs, provavelmente não necessitam de regulação a nível transcripcional. As Rho GTPases atípicas, que estão constitutivamente activas, apresentam um indução global dos pAs distais, distintas das Rho GTPases clássicas. Curiosamente, este padrão sugere que APA é um mecanismo específico do gene. Como 3’UTRs mais longas providenciam mais locais de ligação para microRNA ou proteínas de ligação ao RNA (RBPs), isto sugere que as Rho GTPases atípicas requerem uma regulação mais fina ao nível co-transcriptional, por APA. Adicionalmente, mostramos que a APA é também específica de cada tipo celular, pela análise da expressão do mRNA em outras células da glia (microglia, astrócitos), e em diferentes tipos de neurónios (corticais, estriatais e hipocampais). Nós observamos o mesmo padrão de APA para as Rho GTPases selecionadas em todas as células da glia. No entanto, em neurónios corticais e do estriado, observámos a existência do alongamento do 3’UTR no mRNA da Rac1 durante o crescimento axonal, o que resulta num aumento da quantidade total de proteína. Em resumo, estes resultados indicam, pela primeira vez, que a APA é um mecanismo específico de cada gene e de cada tipo celular. Para além disso, descobrimos uma expressão diferencial de ambas as isoformas da Cdc42 durante a diferenciação dos OLs e do nervo ciático. Durante a diferenciação in vitro de OLs e in vivo do nervo ciático, observámos um aumento do rácio da expressão entre Cdc42 Iso1/Cdc42 Iso2. Mais ainda, a expressão constitutiva de Cdc42 Iso2 em OLs induz um atraso na diferenciação, enquanto a expressão constitutiva da Cdc42 Iso1 induz um aumento das ramificações, sugerindo uma exacerbação do fenótipo de diferenciação. Assim, estas observações sugerem um papel distinto para as diferentes isoformas de Cdc42 durante a diferenciação de OLs. Globalmente, esta tese abre novos caminhos para explorar no futuro, que podem ter um impacto no nosso conhecimento, na regulação do processo de mielinização/remielinização.

Cleavage and polyadenylation are essential pre-mRNA processing reactions maturing the 3’end of almost all protein encoding eukaryotic mRNAs. Analysis of the sequences required for cleavage and polyadenylation in the human melanocortin 4 receptor (MC4R) and the human transcription factors JUNB and JUND pre-mRNAs revealed that, at least for some mammalian genes, 3’end processing of the primary transcript is independent of previously described auxiliary sequence elements located upstream or downstream of the core poly(A) sequences. The analysis of the MC4R poly(A) site, contrary to the current understanding of mammalian poly(A) sites, showed that mutations of the AUUAAA hexamer sequence had no effect on 3’end processing levels while mutations in the short DSE severely reduced cleavage efficiency. The MC4R poly(A) site uses a potent DSE and to direct maximal cleavage efficiency requires only a short upstream adenosine rich sequence. Furthermore, analysis of the endogenous A-rich human JUNB poly(A) signal validated upstream A-rich core sequences as genuine 3’end formation directing sequences in human non-canonical 3’end formation signals. The results show that a minimal human poly(A) site, similar to yeast and plants, can be defined by an adenosine rich sequence adjacent to a U/GU-rich sequence element and a cleavage site. These findings further imply that some human non-canonical poly(A) sites may be recognised via a similar DSE-dependent mechanism and may not require additional auxiliary sequence elements. Finally, results on the analysis of the EDF1 poly(A) signal show that, in a spliced environment, A-rich sequences are also 3’end formation effectors but depend on an competent upstream splicing reaction for efficient definition of the 3’end processing site.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006.
Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

A cell integrates mitogenic signals received at the plasma membrane with intracellular biochemical changes to direct the events of cell division. Oocytes from Xenopus laevis offer a system that allows molecular dissection of pathways controlling cell growth and division in response to extracellular cues. Xenopus oocytes, physiologically arrested in a G2 like state, respond to the hormone progesterone to reinitiate meiosis and mature into a fertilizable egg. Signals received at the oocyte membrane induce translation of dormant maternal mRNAs that not only drive meiotic entry but also maintain the cell cycle arrest in an egg. A major pathway controlling the translation of these mRNAs is cytoplasmic polyadenylation, facilitated by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) through cis-acting elements in their 3'untranslated regions (3'UTRs). Cytoplasmic polyadenylation requires the phosphorylation of serine174 on CPEB by Aurora-A as well as the translation of a hitherto unknown mRNA. The transcript of the RINGO/Spy gene is a putative candidate for this unknown upstream regulator of CPEB function. RINGO/Spy mRNA is translationally repressed in immature oocytes by a ribonucleoprotein (RNP) complex consisting of the repressor Pumilio-2, the putative activator Deleted in Azoospermia-like (DAZl) and embryonic poly A binding protein (ePAB). Progesterone signaling leads to the dissociation of Pumilio-2 from the mRNP and the ensuing RINGO/Spy protein synthesis, in turn, promotes cytoplasmic polyadenylation and oocyte maturation. Pumilio and its associated proteins, such as Drosophila Brain tumor (Brat) and DAZl, in addition to their cytoplasmic roles have ill-defined functions within the nucleus. We detected DAZl within the nucleoli of telomerase-immortalized human retinal pigment epithelial (RPE) cells in interphase and on acrocentric chromosomes during mitosis. DAZl colocalizes with the RNA polymerase I associated Upstream Binding transcription Factor (UBF), most likely through pre-ribosomal RNA and is a likely component of the Nucleolar Organization Region (NOR). Stably knocking down DAZl in RPEs using short hairpin RNAs results in loss of nucleolar segregation, the physiological outcome of which is under investigation. These preliminary findings indicate an additional role for DAZl within the nucleolus, one likely to be independent from cytoplasmic translational control.

A cell integrates mitogenic signals received at the plasma membrane with intracellular biochemical changes to direct the events of cell division. Oocytes from Xenopus laevis offer a system that allows molecular dissection of pathways controlling cell growth and division in response to extracellular cues. Xenopus oocytes, physiologically arrested in a G2 like state, respond to the hormone progesterone to reinitiate meiosis and mature into a fertilizable egg. Signals received at the oocyte membrane induce translation of dormant maternal mRNAs that not only drive meiotic entry but also maintain the cell cycle arrest in an egg. A major pathway controlling the translation of these mRNAs is cytoplasmic polyadenylation, facilitated by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) through cis-acting elements in their 3'untranslated regions (3'UTRs). Cytoplasmic polyadenylation requires the phosphorylation of serine174 on CPEB by Aurora-A as well as the translation of a hitherto unknown mRNA. The transcript of the RINGO/Spy gene is a putative candidate for this unknown upstream regulator of CPEB function. RINGO/Spy mRNA is translationally repressed in immature oocytes by a ribonucleoprotein (RNP) complex consisting of the repressor Pumilio-2, the putative activator Deleted in Azoospermia-like (DAZl) and embryonic poly A binding protein (ePAB). Progesterone signaling leads to the dissociation of Pumilio-2 from the mRNP and the ensuing RINGO/Spy protein synthesis, in turn, promotes cytoplasmic polyadenylation and oocyte maturation. Pumilio and its associated proteins, such as Drosophila Brain tumor (Brat) and DAZl, in addition to their cytoplasmic roles have ill-defined functions within the nucleus. We detected DAZl within the nucleoli of telomerase-immortalized human retinal pigment epithelial (RPE) cells in interphase and on acrocentric chromosomes during mitosis. DAZl colocalizes with the RNA polymerase I associated Upstream Binding transcription Factor (UBF), most likely through pre-ribosomal RNA and is a likely component of the Nucleolar Organization Region (NOR). Stably knocking down DAZl in RPEs using short hairpin RNAs results in loss of nucleolar segregation, the physiological outcome of which is under investigation. These preliminary findings indicate an additional role for DAZl within the nucleolus, one likely to be independent from cytoplasmic translational control.

碩士
國立中興大學
農業生物科技學研究所
88
貳、 英文摘要 Bamboo mosaic potexvirus (BaMV) contains a single-stranded plus-sense genomic RNA with 5 capped and 3 poly(A) tailed structures. The entire nucleotide sequence of isolate O comprising 6366 nt (excluding the 3 poly (A) tail), consists of a 94- and 142-nt untranslated region (UTR) at the 5 end and 3 end, respectively. The secondary structure of the 3 UTR of BaMV RNA was predicted by the computer algorithm STAR and identified by enzymatic and chemical structural probings. The first three stem-loops named A, B, and C, form a colverleaf-like structure designated domain ABC. The fourth stem-loop designated domain D contains a bulge and an internal loop. The fifth stem-loop has the potential to form a pseudoknot structure with at least 13 adenylate residues and is designated domain E. BaMV-O/IL with a deletion at the 3 side of the internal loop of stem D showed only a 2% of coat protein accumulation to that of wild type. It is possible that the loss of coat protein accumulation is related to the loss of the potexviral conserved putative polyadenylation signal AAUAAA. In order to investigate the possible function of the putative polyadenylation AAUAAA motif at the 3 UTR of BaMV RNA, each residue except the first nucleotide of motif was substituted to the other three nucleotides by a PCR technique. Transcripts derived from wild type and 15 single-point substitution mutants were and inoculated into Nicotiana benthamiana protoplasts. The accumulation levels of viral coat protein and RNAs were detected by Western and Northern blots, respectively, after a 48-hour incubation. The specificity of these nucleotides could be analyzed by comparisons of the accumulation levels of coat protein and RNAs with those of wild type. The accumulation levels of minus-sense RNA can be used to indicate the efficiency of the first step of the viral RNA replication. The ratio of plus/minus will be used to indicate the efficiency of the accumulation of genomic RNAs. If the ratio close to that of wild type indicates the efficiency of genomic RNA synthesis is normal and dependent on the accumulation levels of minus-sense RNA. If the ratio was less than that of wild type that would indicate a defect on the efficiency of genomic RNA accumulation. Therefore, we can divide these mutants into four different categories according their accumulation levels of viral products. Two of the mutants have no effect on viral RNA replication. Six of them have effect on viral RNA replication according to the less minus-sense RNA accumulation than that of wild type. However, the accumulation of genomic RNA was dependent on the minus-sense RNA. Another six of them showed not only defect on minus-sense RNA accumulation and but also inefficient of plus-sense genomic RNA accumulation. Only one of them has no effect on minus-sense RNA synthesis but showed less amount of genomic RNA accumulation could be a defect on the efficiency of polyadenylation. Therefore, we conclude that this putative polyadenylation signal at the 3 UTR of BaMV can not only be a proposed polyadenylation signal, but also involved in the minus-sense RNA synthesis.