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Biochimica Et Biophysica Acta Apr 2008Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best... (Review)
Review
Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best studied example is the regulation of translation by cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region of mRNAs involved in Xenopus oocyte maturation. In this review we discuss the mechanism of translational control by the CPE binding protein (CPEB) in Xenopus oocytes as follows: 1. The cytoplasmic polyadenylation machinery such as CPEB, the subunits of cleavage and polyadenylation specificity factor (CPSF), symplekin, Gld-2 and poly(A) polymerase (PAP). 2. The signal transduction that leads to the activation of CPE-mediated polyadenylation during oocyte maturation, including the potential roles of kinases such as MAPK, Aurora A, CamKII, cdk1/Ringo and cdk1/cyclin B. 3. The role of deadenylation and translational repression, including the potential involvement of PARN, CCR4/NOT, maskin, pumilio, Xp54 (Ddx6, Rck), other P-body components and isoforms of the cap binding initiation factor eIF4E. Finally we discuss some of the remaining questions regarding the mechanisms of translational regulation by cytoplasmic polyadenylation and give our view on where our knowledge is likely to be expanded in the near future.
Topics: Animals; Cytoplasm; Female; Oocytes; Oogenesis; Poly A; Polyadenylation; Polynucleotide Adenylyltransferase; Protein Biosynthesis; Protein Kinases; RNA-Binding Proteins; Signal Transduction; Xenopus; Xenopus Proteins
PubMed: 18316045
DOI: 10.1016/j.bbagrm.2008.02.002 -
Nucleic Acids Research Jun 2018RNA 3' polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to...
RNA 3' polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to high levels of the intercalating agent ethidium bromide (EtBr), greater than those required to suppress mitochondrial transcription, mitochondrial tRNAs in human cells became polyadenylated. Relaxation of the inducing stress led to rapid turnover of the polyadenylated tRNAs. The extent, kinetics and duration of tRNA polyadenylation were EtBr dose-dependent, with mitochondrial tRNAs differentially sensitive to the stress. RNA interference and inhibitor studies indicated that ongoing mitochondrial ATP synthesis, plus the mitochondrial poly(A) polymerase and SUV3 helicase were required for tRNA polyadenylation, while polynucleotide phosphorylase counteracted the process and was needed, along with SUV3, for degradation of the polyadenylated tRNAs. Doxycycline treatment inhibited both tRNA polyadenylation and turnover, suggesting a possible involvement of the mitoribosome, although other translational inhibitors had only minor effects. The dysfunctional tRNALeu(UUR) bearing the pathological A3243G mutation was constitutively polyadenylated at a low level, but this was markedly enhanced after doxycycline treatment. We propose that polyadenylation of structurally and functionally abnormal mitochondrial tRNAs entrains their PNPase/SUV3-mediated destruction, and that this pathway could play an important role in mitochondrial diseases associated with tRNA mutations.
Topics: Cell Line, Tumor; DEAD-box RNA Helicases; DNA, Mitochondrial; Ethidium; Humans; Mitochondria; Poly A; Polyadenylation; RNA, Transfer; RNA, Transfer, Leu
PubMed: 29518244
DOI: 10.1093/nar/gky159 -
Genome Biology Oct 2018In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes...
BACKGROUND
In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes in pre-mRNA processing occurring as fibroblasts enter the proliferative cell cycle are also important for promoting their migration.
RESULTS
RNA sequencing of fibroblasts induced into quiescence by contact inhibition reveals downregulation of genes involved in mRNA processing, including splicing and cleavage and polyadenylation factors. These genes also show differential exon use, especially increased intron retention in quiescent fibroblasts compared to proliferating fibroblasts. Mapping the 3' ends of transcripts reveals that longer transcripts from distal polyadenylation sites are more prevalent in quiescent fibroblasts and are associated with increased expression and transcript stabilization based on genome-wide transcript decay analysis. Analysis of dermal excisional wounds in mice reveals that proliferating cells adjacent to wounds express higher levels of cleavage and polyadenylation factors than quiescent fibroblasts in unwounded skin. Quiescent fibroblasts contain reduced levels of the cleavage and polyadenylation factor CstF-64. CstF-64 knockdown recapitulates changes in isoform selection and gene expression associated with quiescence, and results in slower migration.
CONCLUSIONS
Our findings support cleavage and polyadenylation factors as a link between cellular proliferation state and migration.
Topics: Cell Cycle; Cell Movement; Cells, Cultured; Fibroblasts; Humans; Poly A; Polyadenylation; RNA Splicing; Skin; mRNA Cleavage and Polyadenylation Factors
PubMed: 30360761
DOI: 10.1186/s13059-018-1551-9 -
Nucleic Acids Research Aug 2019Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these...
Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L.
Topics: Alternative Splicing; Aminohydrolases; Cell Line, Transformed; Cell Line, Tumor; Colonic Neoplasms; Disease Progression; Formate-Tetrahydrofolate Ligase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein Group C; Humans; Methylenetetrahydrofolate Dehydrogenase (NADP); Multienzyme Complexes; Neoplasms; Poly A; Polyadenylation; RNA Interference; RNA Isoforms
PubMed: 31147722
DOI: 10.1093/nar/gkz461 -
Briefings in Bioinformatics Nov 2021Rhesus macaque is a unique nonhuman primate model for human evolutionary and translational study, but the error-prone gene models critically limit its applications....
Rhesus macaque is a unique nonhuman primate model for human evolutionary and translational study, but the error-prone gene models critically limit its applications. Here, we de novo defined full-length macaque gene models based on single molecule, long-read transcriptome sequencing in four macaque tissues (frontal cortex, cerebellum, heart and testis). Overall, 8 588 227 poly(A)-bearing complementary DNA reads with a mean length of 14 106 nt were generated to compile the backbone of macaque transcripts, with the fine-scale structures further refined by RNA sequencing and cap analysis gene expression sequencing data. In total, 51 605 macaque gene models were accurately defined, covering 89.7% of macaque or 75.7% of human orthologous genes. Based on the full-length gene models, we performed a human-macaque comparative analysis on polyadenylation (PA) regulation. Using macaque and mouse as outgroup species, we identified 79 distal PA events newly originated in humans and found that the strengthening of the distal PA sites, rather than the weakening of the proximal sites, predominantly contributes to the origination of these human-specific isoforms. Notably, these isoforms are selectively constrained in general and contribute to the temporospatially specific reduction of gene expression, through the tinkering of previously existed mechanisms of nuclear retention and microRNA (miRNA) regulation. Overall, the protocol and resource highlight the application of bioinformatics in integrating multilayer genomics data to provide an intact reference for model animal studies, and the isoform switching detected may constitute a hitherto underestimated regulatory layer in shaping the human-specific transcriptome and phenotypic changes.
Topics: 3' Untranslated Regions; Animals; Evolution, Molecular; Gene Expression Profiling; Gene Expression Regulation; Humans; Macaca mulatta; Models, Genetic; Nucleotide Motifs; Organ Specificity; Poly A; Polyadenylation; RNA Isoforms; RNA Transport; RNA, Messenger; Species Specificity; Transcription, Genetic; Transcriptome
PubMed: 33973996
DOI: 10.1093/bib/bbab157 -
Journal of Visualized Experiments : JoVE Oct 2017Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3' untranslated regions (UTRs)...
Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3' untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3'UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3' end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3' end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3' ends in a wide range of conditions, from 10 or fewer cells.
Topics: 3' Untranslated Regions; Animals; Gene Library; Polyadenylation
PubMed: 29053696
DOI: 10.3791/56129 -
RNA (New York, N.Y.) May 2022During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The...
During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding cleavage sites for over 2600 genes. We find that uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in .
Topics: 3' Untranslated Regions; Animals; Giardia lamblia; Mammals; Poly A; Polyadenylation; RNA, Messenger
PubMed: 35110372
DOI: 10.1261/rna.078793.121 -
Cell Systems Jun 2018Alternative polyadenylation (APA) produces from the same gene multiple mature RNAs with varying 3' ends. Although APA is commonly believed to generate beneficial...
Alternative polyadenylation (APA) produces from the same gene multiple mature RNAs with varying 3' ends. Although APA is commonly believed to generate beneficial functional diversity and be adaptive, we hypothesize that most genes have one optimal polyadenylation site and that APA is caused largely by deleterious polyadenylation errors. The error hypothesis, but not the adaptive hypothesis, predicts that, as the expression level of a gene increases, its polyadenylation diversity declines, relative use of the major (presumably optimal) polyadenylation site increases, and that of each minor (presumably nonoptimal) site decreases. It further predicts that the number of polyadenylation signals per gene is smaller than the random expectation and that polyadenylation signals for major but not minor sites are under purifying selection. All of these predictions are confirmed in mammals, suggesting that numerous defective RNAs are produced in normal cells, many phenotypic variations at the molecular level are nonadaptive, and cellular life is noisier than is appreciated.
Topics: 3' Untranslated Regions; Alternative Splicing; Animals; Databases, Genetic; Gene Expression; Humans; Mammals; Poly A; Polyadenylation; RNA, Messenger; Selection, Genetic
PubMed: 29886108
DOI: 10.1016/j.cels.2018.05.007 -
RNA (New York, N.Y.) Nov 2022The polyadenylation signal (PAS) is a key sequence element for 3'-end cleavage and polyadenylation of messenger RNA precursors (pre-mRNAs). This hexanucleotide motif is...
The polyadenylation signal (PAS) is a key sequence element for 3'-end cleavage and polyadenylation of messenger RNA precursors (pre-mRNAs). This hexanucleotide motif is recognized by the mammalian polyadenylation specificity factor (mPSF), consisting of CPSF160, WDR33, CPSF30, and Fip1 subunits. Recent studies have revealed how the AAUAAA PAS, the most frequently observed PAS, is recognized by mPSF. We report here the structure of human mPSF in complex with the AUUAAA PAS, the second most frequently identified PAS. Conformational differences are observed for the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 nucleotides in AAUAAA, while the binding modes of the remaining 4 nt are essentially identical. The 5' phosphate of U2 moves by 2.6 Å and the U2 base is placed near the six-membered ring of A2 in AAUAAA, where it makes two hydrogen bonds with zinc finger 2 (ZF2) of CPSF30, which undergoes conformational changes as well. We also attempted to determine the binding modes of two rare PAS hexamers, AAGAAA and GAUAAA, but did not observe the RNA in the cryo-electron microscopy density. The residues in CPSF30 (ZF2 and ZF3) and WDR33 that recognize PAS are disordered in these two structures.
Topics: Animals; Humans; Polyadenylation; mRNA Cleavage and Polyadenylation Factors; Cleavage And Polyadenylation Specificity Factor; Cryoelectron Microscopy; RNA, Messenger; Protein Binding; RNA Precursors; Mammals; Nucleotides; Poly A
PubMed: 36130077
DOI: 10.1261/rna.079322.122 -
Nucleic Acids Research Dec 2019Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available...
Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3' polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.
Topics: Cell Line, Tumor; Click Chemistry; DNA Barcoding, Taxonomic; Gene Expression Profiling; Humans; In Situ Hybridization, Fluorescence; MCF-7 Cells; Mass Spectrometry; Poly A; Polyadenylation; RNA, Messenger; Sequence Analysis, RNA; Single-Cell Analysis; Spatio-Temporal Analysis
PubMed: 31584096
DOI: 10.1093/nar/gkz852