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International Journal of Molecular... Jan 2018The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation...
The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the -acting elements, -acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA) sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.
Topics: Base Sequence; Cell Line; Gene Expression Regulation; Gene Order; Genes, Reporter; Genetic Vectors; Humans; Mutation; Nucleotide Motifs; Poly A; Polyadenylation; RNA, Messenger; Regulatory Sequences, Nucleic Acid
PubMed: 29342112
DOI: 10.3390/ijms19010279 -
RNA (New York, N.Y.) Mar 2020Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length...
Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length to control protein production. However, the genome-wide relationship of poly(A) tail changes to mRNA translation during vertebrate oocyte maturation is not known. We used Tail-seq and polyribosome analysis to measure poly(A) tail and translational changes during oocyte maturation in We identified large-scale poly(A) and translational changes during oocyte maturation, with poly(A) tail length changes preceding translational changes. Proteins important for completion of the meiotic divisions and early development exhibited increased polyadenylation and translation during oocyte maturation. A family of U-rich sequence elements was enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. We propose that changes in mRNA polyadenylation are a conserved mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of acting sequence elements.
Topics: Animals; Gene Expression Regulation, Developmental; Genome; Oocytes; Oogenesis; Polyadenylation; Protein Biosynthesis; RNA, Messenger; Regulatory Sequences, Nucleic Acid; Xenopus laevis
PubMed: 31896558
DOI: 10.1261/rna.073247.119 -
Nucleic Acids Research Sep 2019Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more...
Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3' end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation and on two other cancer cell lines sorted in microplates. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.
Topics: Benchmarking; Cell Line, Tumor; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Microfluidic Analytical Techniques; Poly A; RNA, Circular; RNA, Messenger; RNA, Ribosomal; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 31216024
DOI: 10.1093/nar/gkz535 -
PloS One 2013Pre-messenger RNA (mRNA) 3'-end cleavage and subsequent polyadenylation strongly regulate gene expression. In comparison with the upstream or downstream motifs,...
Pre-messenger RNA (mRNA) 3'-end cleavage and subsequent polyadenylation strongly regulate gene expression. In comparison with the upstream or downstream motifs, relatively little is known about the feature differences of polyadenylation [poly(A)] sites among major kingdoms. We suspect that the precise poly(A) sites are very selective, and we therefore mapped mRNA poly(A) sites on complete and nearly complete genomes using mRNA sequences available in the National Center for Biotechnology Information (NCBI) Nucleotide database. In this paper, we describe the mRNA nucleotide [i.e., the poly(A) tail attachment position] that is directly in attachment with the poly(A) tail and the pre-mRNA nucleotide [i.e., the poly(A) tail starting position] that corresponds to the first adenosine of the poly(A) tail in the 29 most-mapped species (2 fungi, 2 protists, 18 animals, and 7 plants). The most representative pre-mRNA dinucleotides covering these two positions were UA, CA, and GA in 17, 10, and 2 of the species, respectively. The pre-mRNA nucleotide at the poly(A) tail starting position was typically an adenosine [i.e., A-type poly(A) sites], sometimes a uridine, and occasionally a cytidine or guanosine. The order was U>C>G at the attachment position but A>>U>C≥G at the starting position. However, in comparison with the mRNA nucleotide composition (base composition), the poly(A) tail attachment position selected C over U in plants and both C and G over U in animals, in both A-type and non-A-type poly(A) sites. Animals, dicot plants, and monocot plants had clear differences in C/G ratios at the poly(A) tail attachment position of the non-A-type poly(A) sites. This study of poly(A) site evolution indicated that the two positions within poly(A) sites had distinct nucleotide compositions and were different among kingdoms.
Topics: Animals; Poly A; Polyadenylation; RNA, Messenger; RNA, Plant
PubMed: 24260238
DOI: 10.1371/journal.pone.0079511 -
Nucleic Acids Research Jan 2020Generated by 3' end cleavage and polyadenylation at alternative polyadenylation (poly(A)) sites, alternative terminal exons account for much of the variation between...
Generated by 3' end cleavage and polyadenylation at alternative polyadenylation (poly(A)) sites, alternative terminal exons account for much of the variation between human transcript isoforms. More than a dozen protocols have been developed so far for capturing and sequencing RNA 3' ends from a variety of cell types and species. In previous studies, we have used these data to uncover novel regulatory signals and cell type-specific isoforms. Here we present an update of the PolyASite (https://polyasite.unibas.ch) resource of poly(A) sites, constructed from publicly available human, mouse and worm 3' end sequencing datasets by enforcing uniform quality measures, including the flagging of putative internal priming sites. Through integrated processing of all data, we identified and clustered sites that are closely spaced and share polyadenylation signals, as these are likely the result of stochastic variations in processing. For each cluster, we identified the representative - most frequently processed - site and estimated the relative use in the transcriptome across all samples. We have established a modern web portal for efficient finding, exploration and export of data. Database generation is fully automated, greatly facilitating incorporation of new datasets and the updating of underlying genome resources.
Topics: Animals; Caenorhabditis elegans; Databases, Nucleic Acid; Humans; Mice; Poly A; Polyadenylation; Sequence Analysis, RNA
PubMed: 31617559
DOI: 10.1093/nar/gkz918 -
PloS One 2016The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using...
The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types-Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth.
Topics: 5' Untranslated Regions; Alternative Splicing; Base Sequence; Cell Nucleus; Chlamydomonas reinhardtii; Computational Biology; Gene Expression Regulation, Plant; Genome, Chloroplast; Genome, Mitochondrial; Genome, Plant; High-Throughput Nucleotide Sequencing; Models, Genetic; Molecular Sequence Data; Nucleotide Motifs; Open Reading Frames; Poly A; Polyadenylation; RNA, Messenger; RNA, Plant
PubMed: 26730730
DOI: 10.1371/journal.pone.0146107 -
RNA (New York, N.Y.) Apr 2013Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through... (Review)
Review
Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through binding of processing proteins to the CTD (carboxyl terminal domain) of the largest RNAPII subunit. These interactions are necessary for efficient processing, but whether factor binding to the CTD and delivery to splicing sites is obligatory or facilitatory is unsettled. Capping, addition of an m(7)Gppp residue (cap) to the initial transcribed residue of a pre-mRNA, occurs within seconds. Splicing of pre-mRNA by spliceosomes at particular sites is most likely committed during transcription by the binding of initiating processing factors and ∼50% of the time is completed in mammalian cells before completion of the primary transcript. This fact has led to an outpouring in the literature about "cotranscriptional splicing." However splicing requires several minutes for completion and can take longer. The RNAPII complex moves through very long introns and also through regions dense with alternating exons and introns at an average rate of ∼3 kb per min and is, therefore, not likely detained at each splice site for more than a few seconds, if at all. Cleavage of the primary transcript at the 3' end and polyadenylation occurs within 30 sec or less at recognized polyA sites, and the majority of newly polyadenylated pre-mRNA molecules are much larger than the average mRNA. Finally, it seems quite likely that the nascent RNA most often remains associated with the chromosomal locus being transcribed until processing is complete, possibly acquiring factors related to the transport of the new mRNA to the cytoplasm.
Topics: Animals; History, 20th Century; History, 21st Century; Humans; Molecular Biology; Poly A; RNA Polymerase II; RNA Precursors; RNA Processing, Post-Transcriptional; RNA Splicing
PubMed: 23440351
DOI: 10.1261/rna.038596.113 -
Molecular Systems Biology Sep 2011Genes containing multiple pre-mRNA cleavage and polyadenylation sites, or polyA sites, express mRNA isoforms with variable 3' untranslated regions (UTRs). By systematic...
Genes containing multiple pre-mRNA cleavage and polyadenylation sites, or polyA sites, express mRNA isoforms with variable 3' untranslated regions (UTRs). By systematic analysis of human and mouse transcriptomes, we found that short 3'UTR isoforms are relatively more abundant when genes are highly expressed whereas long 3'UTR isoforms are relatively more abundant when genes are lowly expressed. Reporter assays indicated that polyA site choice can be modulated by transcriptional activity through the gene promoter. Using global and reporter-based nuclear run-on assays, we found that RNA polymerase II is more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, highly expressed genes tend to have a lower level of nucleosome but higher H3K4me3 and H3K36me3 levels at promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that polyA site usage is generally coupled to transcriptional activity, leading to regulation of alternative polyadenylation by transcription.
Topics: 3' Untranslated Regions; Animals; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; HeLa Cells; Histones; Humans; Methylation; Mice; Nucleic Acid Probes; Nucleosomes; Poly A; Polyadenylation; RNA Cleavage; RNA Isoforms; RNA Precursors; RNA, Messenger; Real-Time Polymerase Chain Reaction; Transcription, Genetic; Transcriptome
PubMed: 21952137
DOI: 10.1038/msb.2011.69 -
Plant Physiology Jan 2020Alternative cleavage and polyadenylation (APA) is increasingly recognized as an important regulatory mechanism in eukaryotic gene expression and is dynamically modulated...
Alternative cleavage and polyadenylation (APA) is increasingly recognized as an important regulatory mechanism in eukaryotic gene expression and is dynamically modulated in a developmental, tissue-specific, or environmentally responsive manner. Given the functional importance of APA and the rapid accumulation of APA sites in plants, a comprehensive and easily accessible APA site database is necessary for improved understanding of APA-mediated gene expression regulation. We present a database called PlantAPAdb that catalogs the most comprehensive APA site data derived from sequences from diverse 3' sequencing protocols and biological samples in plants. Currently, PlantAPAdb contains APA sites in six species, ( and ), Arabidopsis (), , , , and APA sites in PlantAPAdb are available for bulk download and can be queried in a Google-like manner. PlantAPAdb provides rich information of the whole-genome APA sites, including genomic locations, heterogeneous cleavage sites, expression levels, and sample information. It also provides comprehensive poly(A) signals for APA sites in different genomic regions according to distinct profiles of cis-elements in plants. In addition, PlantAPAdb contains events of 3' untranslated region shortening/lengthening resulting from APA, which helps to understand the mechanisms underlying systematic changes in 3' untranslated region lengths. Additional information about conservation of APA sites in plants is also available, providing insights into the evolutionary polyadenylation configuration across species. As a user-friendly database, PlantAPAdb is a large and extendable resource for elucidating APA mechanisms, APA conservation, and gene expression regulation.
Topics: Arabidopsis; Chlamydomonas reinhardtii; Genome, Plant; Medicago truncatula; Oryza; Poly A; Polyadenylation; Trifolium
PubMed: 31767692
DOI: 10.1104/pp.19.00943 -
PLoS Genetics Aug 2020Alternative polyadenylation (APA) is emerging as a widespread regulatory layer since the majority of human protein-coding genes contain several polyadenylation (p(A))...
Alternative polyadenylation (APA) is emerging as a widespread regulatory layer since the majority of human protein-coding genes contain several polyadenylation (p(A)) sites in their 3'UTRs. By generating isoforms with different 3'UTR length, APA potentially affects mRNA stability, translation efficiency, nuclear export, and cellular localization. Polyadenylation sites are regulated by adjacent RNA cis-regulatory elements, the principals among them are the polyadenylation signal (PAS) AAUAAA and its main variant AUUAAA, typically located ~20-nt upstream of the p(A) site. Mutations in PAS and other auxiliary poly(A) cis-elements in the 3'UTR of several genes have been shown to cause human Mendelian diseases, and to date, only a few common SNPs that regulate APA were associated with complex diseases. Here, we systematically searched for SNPs that affect gene expression and human traits by modulation of 3'UTR APA. First, focusing on the variants most likely to exert the strongest effect, we identified 2,305 SNPs that interrupt the canonical PAS or its main variant. Implementing pA-QTL tests using GTEx RNA-seq data, we identified 330 PAS SNPs (called PAS pA-QTLs) that were significantly associated with the usage of their p(A) site. As expected, PAS-interrupting alleles were mostly linked with decreased cleavage at their p(A) site and the consequential 3'UTR lengthening. However, interestingly, in ~10% of the cases, the PAS-interrupting allele was associated with increased usage of an upstream p(A) site and 3'UTR shortening. As an indication of the functional effects of these PAS pA-QTLs on gene expression and complex human traits, we observed for few dozens of them marked colocalization with eQTL and/or GWAS signals. The PAS-interrupting alleles linked with 3'UTR lengthening were also strongly associated with decreased gene expression, indicating that shorter isoforms generated by APA are generally more stable than longer ones. Last, we carried out an extended, genome-wide analysis of 3'UTR variants and detected thousands of additional pA-QTLs having weaker effects compared to the PAS pA-QTLs.
Topics: Gene Expression Regulation; Humans; Poly A; Polyadenylation; Polymorphism, Single Nucleotide; RNA 3' Polyadenylation Signals; RNA Stability; RNA, Messenger
PubMed: 32804959
DOI: 10.1371/journal.pgen.1008977