The Experts below are selected from a list of 20910 Experts worldwide ranked by ideXlab platform
Joel D. Richter - One of the best experts on this subject based on the ideXlab platform.
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bidirectional control of mrna translation and synaptic plasticity by the cytoplasmic Polyadenylation complex
Molecular Cell, 2012Co-Authors: Tsuyoshi Udagawa, Sharon A Swanger, Koichi Takeuchi, Jong Heon Kim, Vijayalaxmi C Nalavadi, Jihae Shin, Lori J Lorenz, Suzanne R Zukin, Gary J Bassell, Joel D. RichterAbstract:Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated Polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and Polyadenylation in dendrites. A screen for mRNAs whose Polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for Polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity.
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cpeb and two poly a polymerases control mir 122 stability and p53 mrna translation
Nature, 2011Co-Authors: David M Burns, Andrea Dambrogio, Stephanie Nottrott, Joel D. RichterAbstract:Cytoplasmic Polyadenylation-induced translation controls germ cell development, neuronal synaptic plasticity and cellular senescence, a tumour-suppressor mechanism that limits the replicative lifespan of cells. The cytoplasmic Polyadenylation element binding protein (CPEB) promotes Polyadenylation by nucleating a group of factors including defective in germline development 2 (Gld2), a non-canonical poly(A) polymerase, on specific messenger RNA (mRNA) 3' untranslated regions (UTRs). Because CPEB regulation of p53 mRNA Polyadenylation/translation is necessary for cellular senescence in primary human diploid fibroblasts, we surmised that Gld2 would be the enzyme responsible for poly(A) addition. Here we show that depletion of Gld2 surprisingly promotes rather than inhibits p53 mRNA Polyadenylation/translation, induces premature senescence and enhances the stability of CPEB mRNA. The CPEB 3' UTR contains two miR-122 binding sites, which when deleted, elevate mRNA translation, as does an antagomir of miR-122. Although miR-122 is thought to be liver specific, it is present in primary fibroblasts and destabilized by Gld2 depletion. Gld4, a second non-canonical poly(A) polymerase, was found to regulate p53 mRNA Polyadenylation/translation in a CPEB-dependent manner. Thus, translational regulation of p53 mRNA and cellular senescence is coordinated by Gld2/miR-122/CPEB/Gld4.
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cpeb regulation of human cellular senescence energy metabolism and p53 mrna translation
Genes & Development, 2008Co-Authors: David M Burns, Joel D. RichterAbstract:Cytoplasmic Polyadenylation element-binding protein (CPEB) stimulates Polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic Polyadenylation elements in its 3′ untranslated region (UTR), which promote Polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ∼50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA Polyadenylation-induced translation.
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symplekin and xgld 2 are required for cpeb mediated cytoplasmic Polyadenylation
Cell, 2004Co-Authors: Daron C Barnard, James L. Manley, Kevin Ryan, Joel D. RichterAbstract:Cytoplasmic Polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo Polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous Polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for Polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before Polyadenylation begins. The identification of these factors has broad implications for biological process that employ Polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.
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dissolution of the maskin eif4e complex by cytoplasmic Polyadenylation and poly a binding protein controls cyclin b1 mrna translation and oocyte maturation
The EMBO Journal, 2002Co-Authors: Quiping Cao, Joel D. RichterAbstract:Cytoplasmic Polyadenylation stimulates the translation of several dormant mRNAs during oocyte maturation in Xenopus. Polyadenylation is regulated by the cytoplasmic Polyadenylation element (CPE), a cis-acting element in the 3′-untranslated region of responding mRNAs, and its associated factor CPEB. CPEB also binds maskin, a protein that in turn interacts with eIF4E, the cap-binding factor. Here, we report that based on antibody and mRNA reporter injection assays, maskin prevents oocyte maturation and the translation of the CPE-containing cyclin B1 mRNA by blocking the association of eIF4G with eIF4E. Dissociation of the maskin–eIF4E complex is essential for cyclin B1 mRNA translational activation, and requires not only cytoplasmic Polyadenylation, but also the poly(A)-binding protein. These results suggest a molecular mechanism by which CPE- containing mRNA is activated in early development.
James L. Manley - One of the best experts on this subject based on the ideXlab platform.
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alternative cleavage and Polyadenylation the long and short of it
Trends in Biochemical Sciences, 2013Co-Authors: Bin Tian, James L. ManleyAbstract:Cleavage and Polyadenylation (C/P) of nascent transcripts is essential for maturation of the 3′ ends of most eukaryotic mRNAs. Over the past three decades, biochemical studies have elucidated the machinery responsible for the seemingly simple C/P reaction. Recent genomic analyses have indicated that most eukaryotic genes have multiple cleavage and Polyadenylation sites (pAs), leading to transcript isoforms with different coding potentials and/or variable 3′ untranslated regions (UTRs). As such, alternative cleavage and Polyadenylation (APA) is an important layer of gene regulation impacting mRNA metabolism. Here, we review our current understanding of APA and recent progress in this field.
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transcriptional activators enhance Polyadenylation of mrna precursors
Molecular Cell, 2011Co-Authors: Takashi Nagaike, Charlotte Logan, Ikuko Hotta, Orit Rozenblattrosen, Matthew Meyerson, James L. ManleyAbstract:Summary Polyadenylation of mRNA precursors is frequently coupled to transcription by RNA polymerase II. Although this coupling is known to involve interactions with the C-terminal domain of the RNA polymerase II largest subunit, the possible role of other factors is not known. Here we show that a prototypical transcriptional activator, GAL4-VP16, stimulates transcription-coupled Polyadenylation in vitro. In the absence of GAL4-VP16, specifically initiated transcripts accumulated but little Polyadenylation was observed, while in its presence Polyadenylation was strongly enhanced. We further show that this stimulation requires the transcription elongation-associated PAF complex (PAF1c), as PAF1c depletion blocked GAL4-VP16-stimulated Polyadenylation. Furthermore, knockdown of PAF subunits by siRNA resulted in decreased 3′ cleavage, and nuclear export, of mRNA in vivo. Finally, we show that GAL4-VP16 interacts directly with PAF1c and recruits it to DNA templates. Our results indicate that a transcription activator can stimulate transcription-coupled 3′ processing and does so via interaction with PAF1c.
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symplekin and xgld 2 are required for cpeb mediated cytoplasmic Polyadenylation
Cell, 2004Co-Authors: Daron C Barnard, James L. Manley, Kevin Ryan, Joel D. RichterAbstract:Cytoplasmic Polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo Polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous Polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for Polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before Polyadenylation begins. The identification of these factors has broad implications for biological process that employ Polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.
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evolutionarily conserved interaction between cstf 64 and pc4 links transcription Polyadenylation and termination
Molecular Cell, 2001Co-Authors: Olga Calvo, James L. ManleyAbstract:Abstract Tight connections exist between transcription and subsequent processing of mRNA precursors, and interactions between the transcription and Polyadenylation machineries seem especially extensive. Using a yeast two-hybrid screen to identify factors that interact with the Polyadenylation factor CstF-64, we uncovered an interaction with the transcriptional coactivator PC4. Both human proteins have yeast homologs, Rna15p and Sub1p, respectively, and we show that these two proteins also interact. Given evidence that certain Polyadenylation factors, including Rna15p, are necessary for termination in yeast, we show that deletion or overexpression of SUB1 suppresses or enhances, respectively, both growth and termination defects detected in an rna15 mutant strain. Our findings provide an additional, unexpected connection between transcription and Polyadenylation and suggest that PC4/Sub1p, via its interaction with CstF-64/Rna15p, possesses an evolutionarily conserved antitermination activity.
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complex protein interactions within the human Polyadenylation machinery identify a novel component
Molecular and Cellular Biology, 2000Co-Authors: Yoshio Takagaki, James L. ManleyAbstract:Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of Polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast Polyadenylation machinery. We show that multiple factors, including CstF, cleavage-Polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the Polyadenylation machinery.
Yoshinori Kumazawa - One of the best experts on this subject based on the ideXlab platform.
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Variation and evolution of Polyadenylation profiles in sauropsid mitochondrial mRNAs as deduced from the high-throughput RNA sequencing
BMC Genomics, 2017Co-Authors: Masaki Kurisaki, Yasuyuki Hashiguchi, Yoshinori KumazawaAbstract:Background Genes encoded in vertebrate mitochondrial DNAs are transcribed as a polycistronic transcript for both strands, which is later processed into individual mRNAs, rRNAs and tRNAs, followed by modifications, such as Polyadenylation at the 3′ end of mRNAs. Although mechanisms of the mitochondrial transcription and RNA processing have been extensively studied using some model organisms, structural variability of mitochondrial mRNAs across different groups of vertebrates is poorly understood. We conducted the high-throughput RNA sequencing to identify major Polyadenylation sites for mitochondrial mRNAs in the Japanese grass lizard, Takydromus tachydromoides and compared the Polyadenylation profiles with those identified similarly for 23 tetrapod species, featuring sauropsid taxa (reptiles and birds). Results As compared to the human, a major Polyadenylation site for the NADH dehydrogenase subunit 5 mRNA of the grass lizard was located much closer to its stop codon, resulting in considerable truncation of the 3′ untranslated region for the mRNA. Among the other sauropsid taxa, several distinct Polyadenylation profiles from the human counterpart were found for different mRNAs. They included various truncations of the 3′ untranslated region for NADH dehydrogenase subunit 5 mRNA in four taxa, bird-specific Polyadenylation of the light-strand-transcribed NADH dehydrogenase subunit 6 mRNA, and the combination of the ATP synthase subunit 8/6 mRNA with a neighboring mRNA into a tricistronic mRNA in the side-necked turtle Pelusios castaneus . In the last case of P. castaneus , as well as another example for NADH dehydrogenase subunit 1 mRNAs of some birds, the association between the Polyadenylation site change and the gene overlap was highlighted. The variations in the Polyadenylation profile were suggested to have arisen repeatedly in diverse sauropsid lineages. Some of them likely occurred in response to gene rearrangements in the mitochondrial DNA but the others not. Conclusions These results demonstrate structural variability of mitochondrial mRNAs in sauropsids. The efficient and comprehensive characterization of the mitochondrial mRNAs will contribute to broaden our understanding of their structural and functional evolution.
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variation and evolution of Polyadenylation profiles in sauropsid mitochondrial mrnas as deduced from the high throughput rna sequencing
BMC Genomics, 2017Co-Authors: Yao Sun, Masaki Kurisaki, Yasuyuki Hashiguchi, Yoshinori KumazawaAbstract:Genes encoded in vertebrate mitochondrial DNAs are transcribed as a polycistronic transcript for both strands, which is later processed into individual mRNAs, rRNAs and tRNAs, followed by modifications, such as Polyadenylation at the 3′ end of mRNAs. Although mechanisms of the mitochondrial transcription and RNA processing have been extensively studied using some model organisms, structural variability of mitochondrial mRNAs across different groups of vertebrates is poorly understood. We conducted the high-throughput RNA sequencing to identify major Polyadenylation sites for mitochondrial mRNAs in the Japanese grass lizard, Takydromus tachydromoides and compared the Polyadenylation profiles with those identified similarly for 23 tetrapod species, featuring sauropsid taxa (reptiles and birds). As compared to the human, a major Polyadenylation site for the NADH dehydrogenase subunit 5 mRNA of the grass lizard was located much closer to its stop codon, resulting in considerable truncation of the 3′ untranslated region for the mRNA. Among the other sauropsid taxa, several distinct Polyadenylation profiles from the human counterpart were found for different mRNAs. They included various truncations of the 3′ untranslated region for NADH dehydrogenase subunit 5 mRNA in four taxa, bird-specific Polyadenylation of the light-strand-transcribed NADH dehydrogenase subunit 6 mRNA, and the combination of the ATP synthase subunit 8/6 mRNA with a neighboring mRNA into a tricistronic mRNA in the side-necked turtle Pelusios castaneus. In the last case of P. castaneus, as well as another example for NADH dehydrogenase subunit 1 mRNAs of some birds, the association between the Polyadenylation site change and the gene overlap was highlighted. The variations in the Polyadenylation profile were suggested to have arisen repeatedly in diverse sauropsid lineages. Some of them likely occurred in response to gene rearrangements in the mitochondrial DNA but the others not. These results demonstrate structural variability of mitochondrial mRNAs in sauropsids. The efficient and comprehensive characterization of the mitochondrial mRNAs will contribute to broaden our understanding of their structural and functional evolution.
Daniel Gautheret - One of the best experts on this subject based on the ideXlab platform.
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using alu elements as Polyadenylation sites a case of retroposon exaptation
Molecular Biology and Evolution, 2009Co-Authors: Takeshi Ara, Chongjian Chen, Daniel GautheretAbstract:Of the 1.1 million Alu retroposons in the human genome, about 10,000 are inserted in the 3' untranslated regions (UTR) of protein-coding genes and 1% of these (107 events) are active as Polyadenylation sites (PASs). Strikingly, although Alu's in 3' UTR are indifferently inserted in the forward or reverse direction, 99% of Polyadenylation-active Alu sequences are forward oriented. Consensus Alu+ sequences contain sites that can give rise to Polyadenylation signals and enhancers through a few point mutations. We found that the strand bias of Polyadenylation-active Alu's reflects a radical difference in the fitness of sense and antisense Alu's toward cleavage/Polyadenylation activity. In contrast to previous beliefs, Alu inserts do not necessarily represent weak or cryptic PASs; instead, they often constitute the major or the unique PAS in a gene, adding to the growing list of Alu exaptations. Finally, some Alu-borne PASs are intronic and produce truncated transcripts that may impact gene function and/or contribute to gene remodeling.
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alttrans transcript pattern variants annotated for both alternative splicing and alternative Polyadenylation
BMC Bioinformatics, 2006Co-Authors: Vincent Le Texier, Daniel Gautheret, Fabrice Lopez, Jeanjack M Riethoven, Vasudev Kumanduri, Chellappa Gopalakrishnan, Thangavel Alphonse ThanarajAbstract:The three major mechanisms that regulate transcript formation involve the selection of alternative sites for transcription start (TS), splicing, and Polyadenylation. Currently there are efforts that collect data & annotation individually for each of these variants. It is important to take an integrated view of these data sets and to derive a data set of alternate transcripts along with consolidated annotation. We have been developing in the past computational pipelines that generate value-added data at genome-scale on individual variant types; these include AltSplice on splicing and AltPAS on Polyadenylation. We now extend these pipelines and integrate the resultant data sets to facilitate an integrated view of the contributions from splicing and Polyadenylation in the formation of transcript variants. The AltSplice pipeline examines gene-transcript alignments and delineates alternative splice events and splice patterns; this pipeline is extended as AltTrans to delineate isoform transcript patterns for each of which both introns/exons and 'terminating' polyA site are delineated; EST/mRNA sequences that qualify the transcript pattern confirm both the underlying splicing and Polyadenylation. The AltPAS pipeline examines gene-transcript alignments and delineates all potential polyA sites irrespective of underlying splicing patterns. Resultant polyA sites from both AltTrans and AltPAS are merged. The generated database reports data on alternative splicing, alternative Polyadenylation and the resultant alternate transcript patterns; the basal data is annotated for various biological features. The data (named as integrated AltTrans data) generated for both the organisms of human and mouse is made available through the Alternate Transcript Diversity web site at http://www.ebi.ac.uk/atd/ . The reported data set presents alternate transcript patterns that are annotated for both alternative splicing and alternative Polyadenylation. Results based on current transcriptome data indicate that the contribution of alternative splicing is larger than that of alternative Polyadenylation.
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sequence determinants in human Polyadenylation site selection
BMC Genomics, 2003Co-Authors: Matthieu Legendre, Daniel GautheretAbstract:Background Differential Polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative Polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human Polyadenylation signals in search of patterns that would help discriminate strong and weak Polyadenylation sites, or true sites from randomly occurring signals.
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identification of alternate Polyadenylation sites and analysis of their tissue distribution using est data
Genome Research, 2001Co-Authors: Emmanuel Beaudoing, Daniel GautheretAbstract:Alternate Polyadenylation affects a large fraction of higher eucaryote mRNAs, producing mature transcripts with 3′ ends of variable length. This variation is poorly represented in the current transcript catalogs derived from whole genome sequences, mostly because such posttranscriptional events are not detectable directly at the DNA level. Alternate polydenylation of an mRNA is better understood by comparision to EST databases. Comparing ESTs to mRNAs, however, is a difficult task subjected to the pitfalls of internal priming, presence of intron sequences, repeated elements, chimerical ESTs or matches with EST from paralogous genes. We present here a computer program that addresses these problems and displays ESTs matches to a query mRNA sequence to predict alternate Polyadenylation and to suggest library-specific forms. The output highlights effective Polyadenylation signals, possible sources of artifacts such as A-rich stretches in the mRNA sequences, and allows for a direct visualization of EST libraries using color codes. Statistical biases in the distribution of alternative mRNA forms among EST libraries were systematically sought. About 1450 human and 200 mouse mRNAs displayed such biases, suggesting in each case a tissue- or disease-specific regulation of Polyadenylation.
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patterns of variant Polyadenylation signal usage in human genes
Genome Research, 2000Co-Authors: Emmanuel Beaudoing, Jeanmichel Claverie, Susan M Freier, Jacqueline R Wyatt, Daniel GautheretAbstract:The formation of mature mRNAs in vertebrates involves the cleavage and Polyadenylation of the pre-mRNA, 10-30 nt downstream of an AAUAAA or AUUAAA signal sequence. The extensive cDNA data now available shows that these hexamers are not strictly conserved. In order to identify variant Polyadenylation signals on a large scale, we compared over 8700 human 3' untranslated sequences to 157,775 polyadenylated expressed sequence tags (ESTs), used as markers of actual mRNA 3' ends. About 5600 EST-supported putative mRNA 3' ends were collected and analyzed for significant hexameric sequences. Known Polyadenylation signals were found in only 73% of the 3' fragments. Ten single-base variants of the AAUAAA sequence were identified with a highly significant occurrence rate, potentially representing 14.9% of the actual Polyadenylation signals. Of the mRNAs, 28.6% displayed two or more Polyadenylation sites. In these mRNAs, the poly(A) sites proximal to the coding sequence tend to use variant signals more often, while the 3'-most site tends to use a canonical signal. The average number of ESTs associated with each signal type suggests that variant signals (including the common AUUAAA) are processed less efficiently than the canonical signal and could therefore be selected for regulatory purposes. However, the position of the site in the untranslated region may also play a role in Polyadenylation rate.
Mihaela Zavolan - One of the best experts on this subject based on the ideXlab platform.
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alternative cleavage and Polyadenylation in health and disease
Nature Reviews Genetics, 2019Co-Authors: Andreas J Gruber, Mihaela ZavolanAbstract:Most human genes have multiple sites at which RNA 3' end cleavage and Polyadenylation can occur, enabling the expression of distinct transcript isoforms under different conditions. Novel methods to sequence RNA 3' ends have generated comprehensive catalogues of Polyadenylation (poly(A)) sites; their analysis using innovative computational methods has revealed how poly(A) site choice is regulated by core RNA 3' end processing factors, such as cleavage factor I and cleavage and Polyadenylation specificity factor, as well as by other RNA-binding proteins, particularly splicing factors. Here, we review the experimental and computational methods that have enabled the global mapping of mRNA and of long non-coding RNA 3' ends, quantification of the resulting isoforms and the discovery of regulators of alternative cleavage and Polyadenylation (APA). We highlight the different types of APA-derived isoforms and their functional differences, and illustrate how APA contributes to human diseases, including cancer and haematological, immunological and neurological diseases.
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reconstitution of cpsf active in Polyadenylation recognition of the Polyadenylation signal by wdr33
Genes & Development, 2014Co-Authors: Lars Schönemann, Uwe Kuhn, Andreas Gruber, Georges Martin, Mihaela Zavolan, Walter Keller, Peter Schäfer, Elmar WahleAbstract:Cleavage and Polyadenylation specificity factor (CPSF) is the central component of the 3′ processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the Polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and Polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent Polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the Polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.
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means to an end mechanisms of alternative Polyadenylation of messenger rna precursors
Wiley Interdisciplinary Reviews - Rna, 2014Co-Authors: Andreas Gruber, Georges Martin, Walter Keller, Mihaela ZavolanAbstract:Expression of mature messenger RNAs (mRNAs) requires appropriate transcription initiation and termination, as well as pre-mRNA processing by capping, splicing, cleavage, and Polyadenylation. A core 3′-end processing complex carries out the cleavage and Polyadenylation reactions, but many proteins have been implicated in the selection of Polyadenylation sites among the multiple alternatives that eukaryotic genes typically have. In recent years, high-throughput approaches to map both the 3′-end processing sites as well as the binding sites of proteins that are involved in the selection of cleavage sites and in the processing reactions have been developed. Here, we review these approaches as well as the insights into the mechanisms of Polyadenylation that emerged from genome-wide studies of Polyadenylation across a range of cell types and states. WIREs RNA 2014, 5:183–196. doi: 10.1002/wrna.1206
