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ELife Nov 2022Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that...
Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.
Topics: Humans; Polyadenylation; Nucleotides; RNA Polymerase II; Poly A; Saccharomyces cerevisiae; 3' Untranslated Regions; Transcription, Genetic
PubMed: 36421680
DOI: 10.7554/eLife.83153 -
PloS One 2017RNA-seq reads containing part of the poly(A) tail of transcripts (denoted as poly(A) reads) provide the most direct evidence for the position of poly(A) sites in the...
RNA-seq reads containing part of the poly(A) tail of transcripts (denoted as poly(A) reads) provide the most direct evidence for the position of poly(A) sites in the genome. However, due to reduced coverage of poly(A) tails by reads, poly(A) reads are not routinely identified during RNA-seq mapping. Nevertheless, recent studies for several herpesviruses successfully employed mapping of poly(A) reads to identify herpesvirus poly(A) sites using different strategies and customized programs. To more easily allow such analyses without requiring additional programs, we integrated poly(A) read mapping and prediction of poly(A) sites into our RNA-seq mapping program ContextMap 2. The implemented approach essentially generalizes previously used poly(A) read mapping approaches and combines them with the context-based approach of ContextMap 2 to take into account information provided by other reads aligned to the same location. Poly(A) read mapping using ContextMap 2 was evaluated on real-life data from the ENCODE project and compared against a competing approach based on transcriptome assembly (KLEAT). This showed high positive predictive value for our approach, evidenced also by the presence of poly(A) signals, and considerably lower runtime than KLEAT. Although sensitivity is low for both methods, we show that this is in part due to a high extent of spurious results in the gold standard set derived from RNA-PET data. Sensitivity improves for poly(A) sites of known transcripts or determined with a more specific poly(A) sequencing protocol and increases with read coverage on transcript ends. Finally, we illustrate the usefulness of the approach in a high read coverage scenario by a re-analysis of published data for herpes simplex virus 1. Thus, with current trends towards increasing sequencing depth and read length, poly(A) read mapping will prove to be increasingly useful and can now be performed automatically during RNA-seq mapping with ContextMap 2.
Topics: Animals; Humans; Poly A; RNA, Messenger; Sequence Analysis, RNA; Software; Transcriptome
PubMed: 28135292
DOI: 10.1371/journal.pone.0170914 -
Molekuliarnaia Biologiia 2017Polyadenylation is the non-template addition of adenosine nucleotides at the 3'-end of RNA, which occurs after transcription and generates a poly(A) tail up to 250-300... (Review)
Review
Polyadenylation is the non-template addition of adenosine nucleotides at the 3'-end of RNA, which occurs after transcription and generates a poly(A) tail up to 250-300 nucleotides long. In the first section of our review, we consider the classical process of mRNA 3'-terminus formation, which involves the cleavage of the transcript synthesized by RNA polymerase II and the associated poly(A) tail synthesis by canonical polyadenylate polymerase. Nucleotide sequences needed for mRNA cleavage and poly(A) tail synthesis, in particular the AAUAAA polyadenylation signal, as well as numerous proteins and their complexes involved in mRNA cleavage and polyadenylation, is described in detail. The significance of the poly(A) tail for prolonging mRNA lifetime and stimulating their translation is discussed. Data presented in the second section demonstrate that RNA transcribed by RNA polymerase III from certain SINEs (Short Interspersed Elements) can undergo AAUAAA-dependent polyadenylation. The structural and functional features of RNA polymerase III determine the unusual character of polyadenylation of RNAs synthesized by this enzyme. The history of recent developments in this area of study have been described in greater detail, in particular the discovery of AAUAAA-dependent polyadenylation of RNA synthesized by RNA polymerase III, which has not been discussed previously. Data on AAUAAA-independent polyadenylation catalyzed by noncanonical TRAMP poly(A)-polymerases (Trf4 and Trf5) have been presented in the third section. These enzymes promote rapid degradation of RNAs by adding a short poly(A) tail to them. This mechanism enables the recognition, poly(A)-marking, and elimination of incorrectly folded noncoding transcripts (e.g. ribosomal and transfer RNAs).
Topics: Animals; Humans; Poly A; Polyadenylation; RNA, Messenger
PubMed: 28537233
DOI: 10.7868/S0026898417010189 -
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 -
RNA (New York, N.Y.) Jun 2022Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA...
Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA features that explain differences in translational efficiency (TE), considering the interplay between TE, mRNA poly(A)-tail lengths, microRNAs, and neuronal activation. In neurons and brain tissues, TE correlates with tail length, and a few dozen mRNAs appear to undergo cytoplasmic polyadenylation upon light or chemical stimulation. However, the correlation between TE and tail length is modest, explaining <5% of TE variance, and even this modest relationship diminishes when accounting for other mRNA features. Thus, tail length appears to affect TE only minimally. Accordingly, miRNAs, which accelerate deadenylation of their mRNA targets, primarily influence target mRNA levels, with no detectable effect on either steady-state tail lengths or TE. Larger correlates with TE include codon composition and predicted mRNA folding energy. When combined in a model, the identified correlates explain 38%-45% of TE variance. These results provide a framework for considering the relative impact of factors that contribute to translational control in neurons. They indicate that when examined in bulk, translational control in neurons largely resembles that of other types of post-embryonic cells. Thus, detection of more specialized control might require analyses that can distinguish translation occurring in neuronal processes from that occurring in cell bodies.
Topics: Gene Expression Regulation; MicroRNAs; Neurons; Poly A; Polyadenylation; Protein Biosynthesis; RNA, Messenger
PubMed: 35273099
DOI: 10.1261/rna.079046.121 -
PloS One 2020Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative...
Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative polyadenylation sites determining the 3'-UTR length of gene transcripts have been extensively studied. However, poly(A) lengths demonstrating dynamic and developmental regulation remain largely unexplored. The recently developed NGS-based methods for genome-wide poly(A) profiling have promoted the study of genom-wide poly(A) dynamics. Here we present a straight forward NGS-method for poly(A) profiling, which applies a direct 3'-end adaptor ligation and the template switching for 5'-end adaptor ligation for cDNA library construction. Poly(A) lengths are directly calculated from base call data using a self-developed pipeline pA-finder. The libraries were directly sequenced from the 3'-UTR regions into the followed poly(A) tails, firstly on NextSeq 500 to produce single-end 300-nt reads, demonstrating the method feasibility and that optimization of the fragmented RNA size for cDNA library construction could detecting longer poly (A) tails. We next applied Poly(A)-seq cDNA libraries containing 40-nt and 120-nt poly(A) tail spike-in RNAs on HiSeq X-ten and NovaSeq 6000 to obtain 150-nt and 250-nt pair-end reads. The sequencing profiles of the spike-in RNAs demonstrated both high accuracy and high quality score in reading poly(A) tails. The poly(A) signal bleeding into the 3' adaptor sequence and a sharp decreased quality score at the junction were observed, allowing the modification of pA-finder to remove homopolymeric signal bleeding. We hope that wide applications of Poly(A)-seq help facilitate the study of the development- and disease-related poly(A) dynamics and regulation, and of the recent emerging mixed tailing regulation.
Topics: 3' Untranslated Regions; Gene Library; Genome; Humans; Poly A; Polyadenylation; Sequence Analysis, RNA; Transcriptome
PubMed: 32544193
DOI: 10.1371/journal.pone.0234696 -
RNA Biology Feb 2021La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear... (Review)
Review
La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear La protein and LARP7, mediates binding to the UUU-3'OH termination motif of nascent RNA polymerase III transcripts. We briefly review La and LARP7 activities for RNA 3' end binding and protection from exonucleases before moving to the more recently uncovered poly(A)-related activities of LARP1 and LARP4. Two features shared by LARP1 and LARP4 are direct binding to poly(A) and to the cytoplasmic poly(A)-binding protein (PABP, also known as PABPC1). LARP1, LARP4 and other proteins involved in mRNA translation, deadenylation, and decay, contain PAM2 motifs with variable affinities for the MLLE domain of PABP. We discuss a model in which these PABP-interacting activities contribute to poly(A) pruning of active mRNPs. Evidence that the SARS-CoV-2 RNA virus targets PABP, LARP1, LARP 4 and LARP 4B to control mRNP activity is also briefly reviewed. Recent data suggests that LARP4 opposes deadenylation by stabilizing PABP on mRNA poly(A) tails. Other data suggest that LARP1 can protect mRNA from deadenylation. This is dependent on a PAM2 motif with unique characteristics present in its La-module. Thus, while nuclear La and LARP7 stabilize small RNAs with 3' oligo(U) from decay, LARP1 and LARP4 bind and protect mRNA 3' poly(A) tails from deadenylases through close contact with PABP.: 5'TOP: 5' terminal oligopyrimidine, LaM: La motif, LARP: La-related protein, LARP1: La-related protein 1, MLLE: mademoiselle, NTR: N-terminal region, PABP: cytoplasmic poly(A)-binding protein (PABPC1), Pol III: RNA polymerase III, PAM2: PABP-interacting motif 2, PB: processing body, RRM: RNA recognition motif, SG: stress granule.
Topics: Amino Acid Motifs; Autoantigens; Humans; Phylogeny; Poly A; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; Protein Domains; RNA Stability; RNA, Messenger; RNA, Viral; Ribonucleoproteins; SARS-CoV-2; SS-B Antigen
PubMed: 33522422
DOI: 10.1080/15476286.2020.1868753 -
RNA (New York, N.Y.) Oct 2019Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only...
Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3'-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present , an R package to estimate poly(A) tail length on ONT long-read sequencing data. operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess 's performance across different poly(A) lengths, demonstrating that is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.
Topics: Nanopores; Poly A; Poly T; Polyadenylation; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 31266821
DOI: 10.1261/rna.071332.119 -
Chemistry (Weinheim An Der Bergstrasse,... Jul 2022Poly(A)-binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate...
Poly(A)-binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate mRNA stability and translational potential, and that oligoadenylate-derived PABP ligands can act as effective translational inhibitors with potential applications in pain management. Although extensive research has focused on protein-RNA and protein-protein interactions involving PABPs, further studies are required to examine the ligand specificity of PABP. In this study, we developed a microscale thermophoresis-based assay to probe the interactions between PABP and oligoadenylate analogs containing different chemical modifications. Using this method, we evaluated oligoadenylate analogs modified with nucleobase, ribose, and phosphate moieties to identify modification hotspots. In addition, we determined the susceptibility of the modified oligos to CNOT7 to identify those with the potential for increased cellular stability. Consequently, we selected two enzymatically stable oligoadenylate analogs that inhibit translation in rabbit reticulocyte lysates with a higher potency than a previously reported PABP ligand. We believe that the results presented in this study and the implemented methodology can be capitalized upon in the future development of RNA-based biological tools.
Topics: Animals; Ligands; Poly A; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; RNA; RNA, Messenger; Rabbits; Structure-Activity Relationship
PubMed: 35575378
DOI: 10.1002/chem.202201115 -
International Journal of Molecular... Aug 2023This study evaluated the wound-healing activity of a polymer, Poly(3,4-ethylenedioxythiophene):poly-(styrene sulfonate) (PEDOT: PSS), and determined its mechanism based...
This study evaluated the wound-healing activity of a polymer, Poly(3,4-ethylenedioxythiophene):poly-(styrene sulfonate) (PEDOT: PSS), and determined its mechanism based on angiogenic activity in a full-thickness excision wound model in Spraque Dawley (SD) rats. Administering PEDOT: PSS (1.6) 1.5 ppm at a dose of 50 mg/kg/day significantly improved wound healing in the SD rats on the eleventh day after the incision was created. PEDOT: PSS-treated animals presented no anti-inflammatory skin effects; however, there was an increase in angiogenic behavior. VEGF was found to be significantly elevated in the PEDOT: PSS-treated groups seven days post-incision. However, only a higher concentration of PEDOT: PSS increased TGF-β1 expression within the same time frame. Our results showed that PEDOT: PSS enhances wound healing activity, mainly in terms of its angiogenic effects. In this paper, we describe the highly conductive macromolecular material PEDOT: PSS, which demonstrated accelerated wound-healing activity in the animal incision model. The results will further provide information regarding the application of PEDOT: PSS as a dressing for medical use.
Topics: Animals; Rats; Polystyrenes; Wound Healing; Thiophenes; Skin; Poly A; Surgical Wound
PubMed: 37628719
DOI: 10.3390/ijms241612539