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Briefings in Bioinformatics Jul 2022The poly(A) tail is a dynamic addition to the eukaryotic mRNA and the change in its length plays an essential role in regulating gene expression through affecting...
The poly(A) tail is a dynamic addition to the eukaryotic mRNA and the change in its length plays an essential role in regulating gene expression through affecting nuclear export, mRNA stability and translation. Only recently high-throughput sequencing strategies began to emerge for transcriptome-wide profiling of poly(A) tail length in diverse developmental stages and organisms. However, there is currently no easy-to-use and universal tool for measuring poly(A) tails in sequencing data from different sequencing protocols. Here we established PolyAtailor, a unified and efficient framework, for identifying and analyzing poly(A) tails from PacBio-based long reads or next generation short reads. PolyAtailor provides two core functions for measuring poly(A) tails, namely Tail_map and Tail_scan, which can be used for profiling tails with or without using a reference genome. Particularly, PolyAtailor can identify all potential tails in a read, providing users with detailed information such as tail position, tail length, tail sequence and tail type. Moreover, PolyAtailor integrates rich functions for poly(A) tail and poly(A) site analyses, such as differential poly(A) length analysis, poly(A) site identification and annotation, and statistics and visualization of base composition in tails. We compared PolyAtailor with three latest methods, FLAMAnalysis, FLEPSeq and PAIsoSeqAnalysis, using data from three sequencing protocols in HeLa samples and Arabidopsis. Results show that PolyAtailor is effective in measuring poly(A) tail length and detecting significance of differential poly(A) length, which achieves much higher sensitivity and accuracy than competing methods. PolyAtailor is available at https://github.com/BMILAB/PolyAtailor.
Topics: Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Poly A; Polyadenylation; RNA, Messenger; Sequence Analysis, RNA
PubMed: 35769001
DOI: 10.1093/bib/bbac271 -
Nature Dec 2021
Topics: Agriculture; Animals; Biotechnology; Cattle; Cloning, Molecular; DNA, Complementary; Growth Hormone; History, 20th Century; Humans; Israel; Molecular Biology; Poly A; RNA, Messenger
PubMed: 34907398
DOI: 10.1038/d41586-021-03696-9 -
RNA (New York, N.Y.) Jul 2022The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential...
The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential roles in translational timing and fertilization in early development, but poly(A) tail dynamics have not been fully explored in post-embryonic systems. Here, we examined the landscape and impact of tail length control during macrophage activation. Upon activation, more than 1500 mRNAs, including proinflammatory genes, underwent distinctive changes in tail lengths. Increases in tail length correlated with mRNA levels regardless of transcriptional activity, and many mRNAs that underwent tail extension encode proteins necessary for immune function and post-transcriptional regulation. Strikingly, we found that , whose protein product destabilizes target transcripts, undergoes tail extension. Our analyses indicate that many mRNAs undergoing tail lengthening are, in turn, degraded by elevated levels of ZFP36, constituting a post-transcriptional feedback loop that ensures transient regulation of transcripts integral to macrophage activation. Taken together, this study establishes the complexity, relevance, and widespread nature of poly(A) tail dynamics, and the resulting post-transcriptional regulation during macrophage activation.
Topics: Gene Expression Regulation; Macrophage Activation; Poly A; Polyadenylation; RNA, Messenger
PubMed: 35512831
DOI: 10.1261/rna.078918.121 -
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 -
Journal of Biomolecular Structure &... Jul 2019The homopolynucleotide (homo-oligonucleotide) tracts function as regulatory elements at various stages of mRNAs life cycle. Numerous cellular proteins specifically bind... (Review)
Review
The homopolynucleotide (homo-oligonucleotide) tracts function as regulatory elements at various stages of mRNAs life cycle. Numerous cellular proteins specifically bind to these tracts. Among them are the different poly(A)-binding proteins, poly(C)-binding proteins, multifunctional fragile X mental retardation protein which binds specifically both to poly(G) and poly(U) and others. Molecular mechanisms of regulation of gene expression mediated by homopolynucleotide tracts in RNAs are not fully understood and the structural diversity of these tracts can contribute substantially to this regulation. This review summarizes current knowledge on different forms of homoribopolynucleotides, in particular, neutral and acidic forms of poly(A) and poly(C), and also biological relevance of homoribopolynucleotide (homoribo-oligonucleotide) tracts is discussed. Under physiological conditions, the acidic forms of poly(A) and poly(C) can be induced by proton transfer from acidic amino acids of proteins to adenine and cytosine bases. Finally, we present potential mechanisms for the regulation of some biological processes through the formation of intramolecular poly(A) duplexes.
Topics: Gene Expression Regulation; Humans; Nucleic Acid Conformation; Poly A; Poly C; Poly G; Poly U; Protons
PubMed: 30052138
DOI: 10.1080/07391102.2018.1503972 -
Nature Protocols Sep 2022Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export,... (Review)
Review
Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.
Topics: Animals; DNA, Complementary; Mammals; Mice; Poly A; RNA; RNA, Messenger; Sequence Analysis, RNA; Transcriptome
PubMed: 35831615
DOI: 10.1038/s41596-022-00704-8 -
International Journal of Molecular... Feb 2020The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions...
The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5' and 3' ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or mGpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5' untranslated region (UTR) and 3' UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.
Topics: 3' Untranslated Regions; Animals; Cell-Free System; Cyclization; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Mice; Peptide Chain Initiation, Translational; Poly A; Protein Biosynthesis; RNA Caps; RNA, Messenger
PubMed: 32121426
DOI: 10.3390/ijms21051677 -
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 -
Methods in Enzymology 2021Alternative polyadenylation (APA) is an essential regulatory mechanism for gene expression. The next generation sequencing provides ample opportunity to precisely...
Alternative polyadenylation (APA) is an essential regulatory mechanism for gene expression. The next generation sequencing provides ample opportunity to precisely delineate APA sites genome-wide. Various methods for profiling transcriptome-wide poly(A) sites were developed. By comparing available methods, the ways for adding sequencing adaptors to fit with the Illumina sequencing platform are different. These methods have identified more than 50% genes that undergo APA in eukaryotes. However, due to the unbalanced PCR during library preparation, accurate quantification of poly(A) sites is still a challenge. Here, we describe an updated poly(A) tag sequencing method that incorporates unique molecular identifier (UMI) into the adaptor for removing quantification bias induced by PCR duplicates. Hence, quantification of poly(A) site usages can be achieved by counting UMIs. This protocol, quantifying poly(A) tag sequencing (QPAT-seq), can be finished in 1 day with reduced cost, and is particularly useful for application with a large number of samples.
Topics: Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Poly A; Polyadenylation; Transcriptome
PubMed: 34183134
DOI: 10.1016/bs.mie.2021.04.002 -
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