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Transcriptional activity regulates alternative cleavage and polyadenylation

ABSTRACT: Genes containing multiple polyadenylation (polyA) sites express mRNA isoforms with variable 3’ untranslated regions (3’UTRs). We found that short and long 3’UTR isoforms were relatively more abundant when genes were highly and lowly expressed, respectively, in human and mouse cells. Consistently, upregulated and downregulated genes were more likely to have shortened and lengthened 3’UTRs, respectively, when genes changed expression under different cell conditions or in response to extracellular stimuli. Using nuclear run-on assays, we found that RNA polymerase II (Pol II) was more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, in line with the difference in polyA site usage, highly expressed genes tend to have higher H3K4me3 and H3K36me3 levels but a lower density of nucleosome around promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that the efficiency of 3’ end processing is generally coupled to transcriptional activity, leading to modulation of polyA site choice by transcription and thus connecting transcriptional control with post-transcriptional regulation via pre-mRNA processing.

ORGANISM(S): Mus musculus

PROVIDER: GSE26512 | GEO | 2011/08/15

SECONDARY ACCESSION(S): PRJNA136695

REPOSITORIES: GEO

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Transcriptional activity regulates alternative cleavage and polyadenylation

Project description:Genes containing multiple polyadenylation (polyA) sites express mRNA isoforms with variable 3’ untranslated regions (3’UTRs). We found that short and long 3’UTR isoforms were relatively more abundant when genes were highly and lowly expressed, respectively, in human and mouse cells. Consistently, upregulated and downregulated genes were more likely to have shortened and lengthened 3’UTRs, respectively, when genes changed expression under different cell conditions or in response to extracellular stimuli. Using nuclear run-on assays, we found that RNA polymerase II (Pol II) was more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, in line with the difference in polyA site usage, highly expressed genes tend to have higher H3K4me3 and H3K36me3 levels but a lower density of nucleosome around promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that the efficiency of 3’ end processing is generally coupled to transcriptional activity, leading to modulation of polyA site choice by transcription and thus connecting transcriptional control with post-transcriptional regulation via pre-mRNA processing. Nascent RNA sequencing of C2C12 cell

2011-08-15 | E-GEOD-26512 | biostudies-arrayexpress

m6A-CLIP identified major presence of m6A in last exons

Project description:By adapting UV cross-linking immune precipitation for m6A (m6A-CLIP), we developed several novel genomic approaches with single-nucleotide resolution to accurately locate tens of thousands of m6A residues in human cells and mouse brain mRNAs. We found over 70% of these residues in the last exon with a very sharp rise (six-fold) within 150-400 nucleotides of the last exon, which overlaps the beginning of many 3′ untranslated region (UTRs). The 3′ UTRs contain ~two-thirds of the last exon m6A and 40 to 45% of the total of this base modification in mRNA. Contrary to earlier studies, we found no preference for location of m6A sites in the coding sequences around STOP codons. Many mRNA 3′ UTRs contain multiple polyA sites at least some of which are subject to regulated choices when cells change growth rate or differentiation state. The m6A density is at a peak early in the 3′ UTR and gradually diminishes in the more distal region of the last exon suggesting a possible inhibitory role of the proximal m6A residues to allow polyA choice of more distal polyA sites. This possibility was supported by finding that a main switch from distal to proximal polyA site choice is associated with m6A loss after triple knockdown of the m6A methylase complex. There is a significant overlap of m6A with identified binding sites for Ago complexes that carry microRNAs known to regulate mRNA stability which might possibly represent cooperation in function. With higher accuracy of m6A identification it is now more realistic to engage in highly localized mutagenesis to more definitively identify m6A function.

2015-09-28 | GSE71154 | GEO

Extensive alternative polyadenylation during zebrafish development

Project description:The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3' untranslated regions (3'UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-Seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-Seq reads substantially increased and improved existing 3'UTR annotations, resulting in confidently identified 3'UTRs for more than 78.79% of the annotated protein-coding genes in zebrafish. Most zebrafish genes undergo alternative CPA with more than a thousand genes using different dominant 3'UTRs at different stages. 3'UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3'UTRs are highly expressed in the ovary yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3'UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At two hours post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3'UTRs provide a resource for studying gene regulation during vertebrate development. 3P-Seq was used to map the 3' ends of protein-coding genes in the zebrafish genome

2012-06-17 | E-GEOD-37453 | biostudies-arrayexpress

Localization of functionally distinct CDC42 isoforms in neurons requires alternative 3'UTR usage

Project description:The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs – alternative splicing and polyadenylation – have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3’UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

2018-12-07 | PXD011518 | Pride

Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products

Project description:Alternative polyadenylation (APA) produces transcript 3’ untranslated regions (3’UTRs) with distinct sequences, lengths, stability, and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3’UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3’UTR products of APA, leading to their systematic downregulation. Further, we find that many APA events consistently observed in multiple tumor types are controlled by NMD. Additionally, PTBP1, previously implicated in direct modulation of co-transcriptional polyA site choice, regulates the balance of short and long 3’UTR isoforms by inhibiting NMD. Our data suggest that PTBP1 binding near polyA sites can drive production of long 3’UTR APA products in the nucleus and/or protect them from decay in the cytoplasm. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

2020-05-28 | GSE138736 | GEO

Extensive alternative polyadenylation during zebrafish development

2012-06-17 | GSE37453 | GEO

CPEB inducible knock-down in a H.D. Myz cell line

Project description:Messenger RNA stability, localization, and translation are largely determined by sequences in their 3′ untranslated regions (3’UTRs), which recruit regulatory proteins and RNAs. More than half of the mammalian genes generate multiple mRNA isoforms differing in their 3′UTRs and therefore in their regulatory elements. The Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1) binds to cognate sites in 3’ UTRs and regulates translation. CPEB1 can shuttle to the nucleus and we report its co-localization with splicing factors. CPEB1 knock down leads to changes in alternative splicing, and we show that alternative 3’ splice site linked to alternative polyadenylation of the bub-3 pre-mRNA, important for cell proliferation, is regulated by CPEB1 at least in part by preventing 3’ splice site recognition by U2AF. RNA-Seq experiments reveal that CPEB1 mediates 3’ UTR shortening of hundreds of mRNAs, leading to changes in their translation efficiency.

2013-02-25 | GSE32440 | GEO

Choice of alternative polyadenylation sites, mediated by the RNA-binding protein Elavl3, plays a role in differentiation of inhibitory neuronal progenitors

Project description:Alternative polyadenylation is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3' UTR and changes of the post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During neuronal differentiation a variety of mRNAs change the length of their 3' UTR, promoting the longer version of the transcripts. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3’ UTRs during differentiation. Enriched expression of the long 3’ UTR variants of Pes1 and Gng2 was detected in areas of cortical and subcortical neuronal differentiation, respectively, in the mouse brain by two-probes FISH analyses. In Drosophila the choice of polyA+ site has been shown to be regulated by the RNA-binding protein Elav, which inhibits polyadenylation at proximal sites while interacting with paused Pol-II promoters. Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. The silencing of Elavl3 in ANS cells resulted in impaired elongation of the 3’UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3’ UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation.

2019-01-10 | GSE119073 | GEO

To investigate the decay constants (half-lives) of transcript isoforms generated by alternative polyadenylation in proliferating and quiescent cells

Project description:Purpose: Alternative polyadenylation (APA) can result in the generation of transcripts that terminate at different locations within the gene, which can result in different 3' UTRs. 3´ UTRs are known to contain RNA stabilizing and destabilizing motifs that provide a platform for binding of RNA binding proteins. Changes in 3´ UTRs for the different APA-generated isoforms of a gene could allow for inclusion or exclusion of these RNA stability elements. The goal of this study is determine the stability (half-lives based on transcript decay) of APA isoforms in proliferating and quiescent cells. Methods: The proliferating and quiescent (7-day contact inhibited) cells were treated with actinomycin D to stop transcription and the cells were harvested at different time points (0. 2, 4, 8 hours) for RNA isolation and global sequencing of 3´ ends. The adaptors and polyA sequenced were trimmed from each read. The reads were aligned to the human genome (hg19) using TopHat (v2.0.14). The aligned read files were used to determine the counts of APA isoforms of each gene using the code provided by Gruber et al. (PMID: 27382025). The counts of the APA isoforms were fit to an exponential decay model to obtain half-lives. The entire workflow was performed for two biological replicates: 12-1 and 12-3 strains of human dermal fibroblasts. Results: In two different fibroblast strains (12-1 and 12-3), we found that isoforms terminating at distal polyadenylation sites were more stable than isoforms terminating at proximal polyadenylation sites in quiescent, but not proliferating, fibroblasts. Conclusions: The transition from shorter to longer isoforms in quiescent cells is associated with stabilization of the transcripts.

2018-08-28 | GSE117121 | GEO

H3K4me3 recognition by the COMPASS complex guides de novo histone methylation to restore the symmetric distribution of H3K4me3 following DNA replication

Project description:During DNA replication, histones in nucleosomes ahead of DNA replication forks are evicted and then distributed equally onto leading and lagging strands. Mcm2, a subunit of the replicative MCM helicase, participates in the transfer of parental H3-H4 marked by H3K4me3 to lagging strands. Mutations in Mcm2 (Mcm2-3A) result in enrichment of H3K4me3 at leading strands following DNA replication. Here, we show that mcm2-3A mutant cells partially recover the H3K4me3 lost at lagging strands, with a faster recovery at highly transcribed genes than lowly ones, before mitosis. Furthermore, the H3K4 methyltransferase complex COMPASS is essential in the recovery of H3K4me3, irrespective of transcription status. Finally, H3K4me3 recognition (read) by COMPASS is also important for the restoration (write) of this mark. We suggest that both gene transcription and the “read and write” mechanism contribute to the restoration of H3K4me3, with the later mechanism more important for lowly transcribed chromatin.

2022-04-13 | GSE184188 | GEO

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