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International Journal of Biological... 2018Alternative polyadenylation is an essential RNA processing event that contributes significantly to regulation of transcriptome diversity and functional dynamics in both... (Review)
Review
Alternative polyadenylation is an essential RNA processing event that contributes significantly to regulation of transcriptome diversity and functional dynamics in both animals and plants. Here we review newly developed next generation sequencing methods for genome-wide profiling of alternative polyadenylation (APA) sites, bioinformatics pipelines for data processing and both wet and dry laboratory approaches for APA validation. The library construction methods LITE-Seq (Low-Input 3'-Terminal sequencing) and PAC-seq (PolyA Click sequencing) tag polyA cDNA, while BAT-seq (BArcoded, three-prime specific sequencing) and PAPERCLIP (oly() binding rotein-mediated mRNA 3'nd etrieval by rossinking mmunorecipitation) enrich polyA RNA. Interestingly, only WTTS-seq (Whole Transcriptome Termini Site sequencing) targets both polyA RNA and polyA cDNA. Varieties of bioinformatics pipelines are well established to pursue read quality control, mapping, clustering, characterization and pathway analysis. The RHAPA (RNase H alternative polyadenylation assay) and 3'RACE-seq (3' rapid amplification of cDNA end sequencing) methods directly validate APA sites, while WTSS-seq (whole transcriptome start site sequencing), RNA-seq (RNA sequencing) and public APA databases can serve as indirect validation methods. We hope that these tools, pipelines and resources trigger huge waves of interest in the research community to investigate APA events underlying physiological, pathological and psychological changes and thus understand the information transfer events from genome to phenome relevant to economically important traits in both animals and plants.
Topics: 3' Untranslated Regions; Animals; DNA, Complementary; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Plants; Poly A; Polyadenylation; RNA, Messenger; Sequence Analysis, RNA; Transcriptome
PubMed: 30416385
DOI: 10.7150/ijbs.27168 -
Proceedings of the National Academy of... Dec 2022Alternative polyadenylation (APA) plays an important role in posttranscriptional gene regulation such as transcript stability and translation efficiency. However, our...
Alternative polyadenylation (APA) plays an important role in posttranscriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at the single-cell level is largely unexplored. Here, we developed single-cell polyadenylation sequencing, a strand-specific approach for sequencing the 3' end of transcripts, to investigate the landscape of APA at the single-cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle genes were significantly enriched in genes with high variation in polyA site usages. Furthermore, the 414 genes showing a polyA site usage switch after cell synchronization enriched cell cycle genes, while the differentially expressed genes after cell synchronization did not enrich cell cycle genes. We further identified 812 genes showing polyA site usage changes between neighboring cell cycles, which were grouped into six clusters, with cell phase-specific functional categories enriched in each cluster. Deletion of one polyA site in and results in slower and faster cell cycle progression, respectively, supporting polyA site usage switch played an important role in cell cycle. These results indicate that APA is an important layer for cell cycle regulation.
Topics: Polyadenylation; Poly A; Genes, cdc; Cell Cycle; Cell Division
PubMed: 36454750
DOI: 10.1073/pnas.2113504119 -
Wiley Interdisciplinary Reviews. RNA 2012Pre-mRNA cleavage and polyadenylation is an essential step for 3' end formation of almost all protein-coding transcripts in eukaryotes. The reaction, involving cleavage... (Review)
Review
Pre-mRNA cleavage and polyadenylation is an essential step for 3' end formation of almost all protein-coding transcripts in eukaryotes. The reaction, involving cleavage of nascent mRNA followed by addition of a polyadenylate or poly(A) tail, is controlled by cis-acting elements in the pre-mRNA surrounding the cleavage site. Experimental and bioinformatic studies in the past three decades have elucidated conserved and divergent elements across eukaryotes, from yeast to human. Here we review histories and current models of these elements in a broad range of species.
Topics: Animals; Computational Biology; Humans; Plants; Poly A; Polyadenylation; Polynucleotide Adenylyltransferase; RNA Precursors; Regulatory Sequences, Nucleic Acid; Saccharomyces cerevisiae; mRNA Cleavage and Polyadenylation Factors
PubMed: 22012871
DOI: 10.1002/wrna.116 -
Wiley Interdisciplinary Reviews. RNA Jan 2023The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a... (Review)
Review
The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a posttranscriptional process that occurs in the nucleus by canonical poly(A) polymerases (PAPs). In some specific instances, the poly(A) tail can also be extended in the cytoplasm by noncanonical poly(A) polymerases (ncPAPs). This epitranscriptomic regulation of mRNA recently became one of the most interesting aspects in the field. Advances in RNA sequencing technologies and software development have allowed the precise measurement of poly(A) tails, identification of new ncPAPs, expansion of the function of known enzymes, discovery and a better understanding of the physiological role of tail heterogeneity, and recognition of a correlation between tail length and RNA translatability. Here, we summarize the development of polyadenylation research methods, including classic low-throughput approaches, Illumina-based genome-wide analysis, and advanced state-of-art techniques that utilize long-read third-generation sequencing with Pacific Biosciences and Oxford Nanopore Technologies platforms. A boost in technical opportunities over recent decades has allowed a better understanding of the regulation of gene expression at the mRNA level. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico.
Topics: Polyadenylation; RNA, Messenger; Cytoplasm; Sequence Analysis, RNA; Cell Nucleus; Poly A
PubMed: 35617484
DOI: 10.1002/wrna.1737 -
Journal of Experimental & Clinical... Feb 2021Occurring in over 60% of human genes, alternative polyadenylation (APA) results in numerous transcripts with differing 3'ends, thus greatly expanding the diversity of... (Review)
Review
Occurring in over 60% of human genes, alternative polyadenylation (APA) results in numerous transcripts with differing 3'ends, thus greatly expanding the diversity of mRNAs and of proteins derived from a single gene. As a key molecular mechanism, APA is involved in various gene regulation steps including mRNA maturation, mRNA stability, cellular RNA decay, and protein diversification. APA is frequently dysregulated in cancers leading to changes in oncogenes and tumor suppressor gene expressions. Recent studies have revealed various APA regulatory mechanisms that promote the development and progression of a number of human diseases, including cancer. Here, we provide an overview of four types of APA and their impacts on gene regulation. We focus particularly on the interaction of APA with microRNAs, RNA binding proteins and other related factors, the core pre-mRNA 3'end processing complex, and 3'UTR length change. We also describe next-generation sequencing methods and computational tools for use in poly(A) signal detection and APA repositories and databases. Finally, we summarize the current understanding of APA in cancer and provide our vision for future APA related research.
Topics: 3' Untranslated Regions; Animals; Computational Biology; Databases, Genetic; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Poly A; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; RNA-Binding Proteins; Signal Transduction
PubMed: 33526057
DOI: 10.1186/s13046-021-01852-7 -
Nature Structural & Molecular Biology Feb 2023Poly(A)-tail-mediated post-transcriptional regulation of maternal mRNAs is vital in the oocyte-to-embryo transition (OET). Nothing is known about poly(A) tail dynamics...
Poly(A)-tail-mediated post-transcriptional regulation of maternal mRNAs is vital in the oocyte-to-embryo transition (OET). Nothing is known about poly(A) tail dynamics during the human OET. Here, we show that poly(A) tail length and internal non-A residues are highly dynamic during the human OET, using poly(A)-inclusive RNA isoform sequencing (PAIso-seq). Unexpectedly, maternal mRNAs undergo global remodeling: after deadenylation or partial degradation into 3'-UTRs, they are re-polyadenylated to produce polyadenylated degradation intermediates, coinciding with massive incorporation of non-A residues, particularly internal long consecutive U residues, into the newly synthesized poly(A) tails. Moreover, TUT4 and TUT7 contribute to the incorporation of these U residues, BTG4-mediated deadenylation produces substrates for maternal mRNA re-polyadenylation, and TENT4A and TENT4B incorporate internal G residues. The maternal mRNA remodeling is further confirmed using PAIso-seq2. Importantly, maternal mRNA remodeling is essential for the first cleavage of human embryos. Together, these findings broaden our understanding of the post-transcriptional regulation of maternal mRNAs during the human OET.
Topics: Humans; RNA, Messenger, Stored; Oocytes; RNA, Messenger; Gene Expression Regulation; Polyadenylation; Poly A
PubMed: 36646905
DOI: 10.1038/s41594-022-00908-2 -
Methods in Enzymology 2021Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is...
Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is under constant regulation by cap-binding proteins, miRNAs, and RNA binding proteins, including poly(A) binding proteins (PABPs). The interplay between all these factors determines whether nuclear or cytoplasmic exoribonucleases will gain access to and remove the poly(A) tail, which is so critical to the stability and translation capacity of the mRNA. In this chapter, we present an overview of two of the key features of the mRNA life-cycle: cleavage/polyadenylation and deadenylation, and describe biochemical assays that have been generated to study the activity of each of these enzymatic reactions. Finally, we also provide protocols to investigate mRNA's poly(A) length. The importance of these assays is highlighted by the dynamic and essential role the poly(A) tail length plays in controlling gene expression.
Topics: Exoribonucleases; Poly A; Polyadenylation; RNA, Messenger; RNA-Binding Proteins
PubMed: 34183126
DOI: 10.1016/bs.mie.2021.04.005 -
Wiley Interdisciplinary Reviews. RNA 2011Degradation of messenger RNAs (mRNAs) plays an essential role in modulation of gene expression and in quality control of mRNA biogenesis. Nearly all major mRNA decay... (Review)
Review
Degradation of messenger RNAs (mRNAs) plays an essential role in modulation of gene expression and in quality control of mRNA biogenesis. Nearly all major mRNA decay pathways characterized thus far in eukaryotes are initiated by deadenylation, i.e., shortening of the mRNA 3(') poly(A) tail. Deadenylation is often a rate-limiting step for mRNA degradation and translational silencing, making it an important control point for both processes. In this review, we discuss the fundamental principles that govern mRNA deadenylation in eukaryotes. We use several major mRNA decay pathways in mammalian cells to illustrate mechanisms and regulation of deadenylation-dependent mRNA decay, including decay directed by adenine/uridine-rich elements (AREs) in the 3(') -untranslated region (UTR), the rapid decay mediated by destabilizing elements in protein-coding regions, the surveillance mechanism that detects and degrades nonsense-containing mRNA [i.e., nonsense-mediated decay (NMD)], the decay directed by miRNAs, and the default decay pathway for stable messages. Mammalian mRNA deadenylation involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. Decapping takes place after deadenylation and may serve as a backup mechanism to trigger mRNA decay if initial deadenylation is compromised. In addition, we discuss how deadenylation impacts the dynamics of RNA processing bodies (P-bodies), where nontranslatable mRNAs can be degraded or stored. Possible models for mechanisms of various deadenylation-dependent mRNA decay pathways are also discussed.
Topics: Animals; Humans; Kinetics; Models, Biological; Poly A; Polyadenylation; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; Signal Transduction
PubMed: 21957004
DOI: 10.1002/wrna.40 -
RNA Biology 2015Transcription initiation and mRNA maturation were long considered co-occurring but separately regulated events of gene control. In the past decade, gene promoters, the... (Review)
Review
Transcription initiation and mRNA maturation were long considered co-occurring but separately regulated events of gene control. In the past decade, gene promoters, the platforms of transcription initiation, have been assigned additional functions such as the regulation of splicing and 3' end processing. In a recent study, Oktaba and Zhang and al. reveal that neural 3' UTR extension is dependent on promoter sequences. In Drosophila neurons, promoter regions of a subset of genes recruit the RNA-binding protein ELAV, which is required for subsequent ELAV-mediated alternative polyadenylation. Intriguingly, RNA Polymerase II pausing at promoters seems to facilitate ELAV recruitment. How transcription initiation and alternative polyadenylation, processes separated by an entire gene length, are functionally linked, remains unsolved. In this article, I summarize recent findings and discuss possible mechanisms.
Topics: 3' Untranslated Regions; Animals; ELAV Proteins; Gene Expression Regulation; Humans; Neurons; Poly A; Polyadenylation; Promoter Regions, Genetic; RNA Polymerase II; Transcription Elongation, Genetic; Transcription Initiation, Genetic
PubMed: 26158379
DOI: 10.1080/15476286.2015.1060393 -
Wiley Interdisciplinary Reviews. RNA 2010Changing the position of the poly(A) tail in an mRNA--alternative polyadenylation--is an important mechanism to increase the diversity of gene expression, especially in... (Review)
Review
Changing the position of the poly(A) tail in an mRNA--alternative polyadenylation--is an important mechanism to increase the diversity of gene expression, especially in metazoans. Alternative polyadenylation often occurs in a tissue- or developmental stage-specific manner and can significantly affect gene activity by changing the protein product generated, the stability of the transcript, its localization, or its translatability. Despite the important regulatory effects that alternative polyadenylation have on gene expression, only a sparse few examples have been mechanistically characterized. Here, we review the known mechanisms for the control of alternative polyadenylation, catalog the tissues that demonstrate a propensity for alternative polyadenylation, and focus on the proteins that are known to regulate alternative polyadenylation in specific tissues. We conclude that the field of alternative polyadenylation remains in its infancy, with possibilities for future investigation on the horizon. Given the profound effect alternative polyadenylation can have on gene expression and human health, improved understanding of alternative polyadenylation could lead to numerous advances in control of gene activity.
Topics: Alternative Splicing; Animals; Brain; Humans; Male; Models, Biological; Organ Specificity; Polyadenylation; Spermatogenesis; Testis
PubMed: 21956945
DOI: 10.1002/wrna.29