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CPSF controls PAS selection and epidermal differentiation

ABSTRACT: Alternative polyadenylation (APA) is a regulatory mechanism that controls gene expression level and function through the usage of different transcription termination sites, but how APA is regulated to influence epidermal tissue differentiation remains under-characterized. Leveraging 3’READS+, we identified prevalent APA in keratinocytes. We further identified a high-confidence list of genes that alters their usages of polyadenylation site (PAS) during keratinocyte differentiation. To begin to understand the mechanisms that modulates PAS selection within a given gene, we identified that the expression of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex is down regulated during keratinocyte differentiation. Suppression of CPSF using RNAi or CRISPRi strongly impaired epidermal regeneration with upregulation of terminal differentiation marker gene expression. Mechanistically, we identified that CPSF suppression decreased the usage of the GRHL3 proximal PAS and upregulated full-length GRHL3 mRNA production. Knockdown of GRHL3 in the context of CPSFi partially restored differentiation marker gene expression. We also found CPSF interacts with RNA-binding proteins to control PAS usage. Thus, our data suggest a model where CPSF binding preference and PAS choices regulate epidermal terminal differentiation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE127223 | GEO | 2020/11/02

REPOSITORIES: GEO

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CPSF controls PAS selection and epidermal differentiation

Project description:CPSF controls PAS selection and epidermal differentiation

| PRJNA524364 | ENA

RNA polyadenylation landscape in human and mouse cells

Project description:Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. We first analyzed HeLa cell transcriptome using PAS-Seq to demonstrate that PAS-Seq not only accurately and comprehensively identifies poly(A) junctions, but also provide quantitative information on the relativel abundance of APA isoforms. Next we analyzed the mouse embryonic stem cells, neural stem/progenitor cells, and neurons by PAS-Seq to characterize the dynamic changes of mRNA polyadenylation during stem cell differentiation.

2011-02-26 | E-GEOD-25450 | biostudies-arrayexpress

Regulation of alternative cleavage and polyadenylation by 3’ end processing and splicing factors

Project description:Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage and polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1, and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly influencing C/P events in 5’ introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells

2015-04-01 | E-GEOD-62001 | biostudies-arrayexpress

Analysis of alternative cleavage and polyadenylation by 3' region extraction and deep sequencing

Project description:Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3' region extraction and deep sequencing (3'READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3' ends revealed over 5,000 previously overlooked pAs (~8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3'READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3' endM-bM-^@M-^Sprocessing activity in development and differentiation. 3'READS to map pAs in mouse genome

2012-12-17 | E-GEOD-42398 | biostudies-arrayexpress

The transcriptional terminator XRN2 and the RNA binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Project description:Alternative cleavage and polyadenylation (APA) of pre-mRNAs yields multiple transcripts differing in their 3' end and its regulation is often aberrant in human cancers, including prostate cancer (PC). In this study, we have uncovered a novel mechanism of APA regulation which impinges on the functional interaction between the 5'-3' exonuclease XRN2 and the A/U-rich motif RNA binding protein Sam68 in PC cells. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor (CPSF) for the recognition of the AAUAAA core polyadenylation signal (PAS) motif. In particular, the Sam68/XRN2 complex suppresses the usage of strong canonical PASs at distal ends of genes and, consequently, promotes the usage of suboptimal proximal PASs.

2022-08-06 | GSE198872 | GEO

Regulation of alternative cleavage and polyadenylation by 3’ end processing and splicing factors

2015-04-01 | GSE62001 | GEO

SRSF7 and SRSF3 affectSRSF7 and SRSF3 affect 3’UTR length in opposite directions by controlling proximal poly(A)-site usage and CFIm levels 3’UTR length in opposite directions by controlling proximal poly(A)-site usage and CFIm levels

Project description:Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which affects the length of their 3’ untranslated regions (3’UTRs). APA regulates stage- and tissue-specific gene expression by affecting the stability, subcellular localization or translation rate of transcripts. We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, link APA to mRNA export. However, the underlying mechanism for APA regulation by SRSF3 and SRSF7 remained unknown. Here, we combined iCLIP and 3’-end sequencing to find that both proteins bind upstream of proximal PAS (pPAS), but exert opposing effects on 3’UTR length. We show that SRSF7 enhances pPAS usage in a splicing-independent and concentration-dependent manner by recruiting the cleavage factor FIP1, thereby generating short 3’UTRs. SRSF7-specific domains that are absent in SRSF3 are necessary and sufficient for FIP1 recruitment. SRSF3 promotes long 3’UTRs by maintaining high levels of the cleavage factor Im (CFIm) via alternative splicing. Using iCLIP, we show that CFIm binds before and after the pPASs of SRSF3 targets, which masks them and inhibits polyadenylation. In the absence of SRSF3, CFIm levels are strongly reduced, which exposes the pPASs and leads to shorter 3’UTRs. Conversely, during cellular differentiation, 3’UTRs are massively extended, while the levels of SRSF7 and FIP1 strongly decline. Altogether, our data suggest that SRSF7 acts as a sequence-specific enhancer of pPASs, while SRSF3 inhibits pPAS usage by controlling CFIm levels. Our data shed light on a long-standing puzzle of how one factor (CFIm) can inhibit and enhance PAS usage.

2021-09-09 | PXD018090 | Pride

A compendium of conserved cleavage and polyadenylation events in mammalian genes

Project description:Cleavage and polyadenylation is essential for 3’ end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3’UTRs and/or coding sequences. Here we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on ~1.2 billion 3’ end sequencing reads from over 360 human, mouse and rat samples. We show that ~80% of mammalian mRNA genes contain at least one conserved PAS, and ~50% have conserved APA events. PAS conservation generally reduces promiscuous 3’ end processing, stabling gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with APA events. While tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. We show enrichment of mRNA destabilizing motifs in alternative 3’UTR sequences, leading to substantial differences in mRNA stability between 3’UTR APA isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Mutations of the U-rich motif around the PAS often accompany APA profile changes between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation involving evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic understanding of APA functions.

2018-08-08 | GSE111134 | GEO

GRHL3 chromatin binding and the super-enhancer landscape are reorganized in different functional states of epidermal keratinocytes

Project description:While the genomic mechanisms underlying progressive, irreversible cell lineage commitments are well-studied, we know little about the chromatin changes during transient cell states such as cell migration. Interestingly, a large number of SEs in NHEK-D and NHEK-M overlap genes encoding transcription factors with important roles in promotion of epidermal differentiation, including GRHL3, TP63, RUNX1, NOTCH3 and FOS. To test the role of these SE-associated transcription factors in a systematic manner, and to place GRHL3 in the context of other keratinocyte differentiation regulators, we used siRNAs to individually knock down GRHL3 and 50 other transcriptional regulators in NHEK-D. These transcriptional regulators were selected based on expression changes during human keratinocyte differentiation. Many have been previously implicated in epidermal differentiation in mice and humans; the genes encoding 22 of them are associated with SEs. To assess the effect of the knockdowns on keratinocyte differentiation, we monitored the expression of approximately 14,000 genes with custom-made Agilent microarrays. Among the 14,000 genes whose expression we monitored were all genes expressed in human keratinocytes and all transcriptional regulators. This 51 x 14,000 gene expression matrix provided a rich dataset to explore gene regulatory networks in epidermal differentiation.

2017-04-12 | GSE86193 | GEO

3âREADS+, a sensitive and accurate method for 3â end sequencing of polyadenylated RNA

Project description:Sequencing of the 3â end of poly(A)+ RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3â region extraction and deep sequencing (3âREADS), to address mispriming issues that often plague 3â end sequencing. Here we report a new version, named 3âREADS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)+ RNA and to remove bulk of the poly(A) tail, 3âREADS+ generates RNA fragments with an optimal number of terminal As that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3âREADS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes, and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (~50%). 3âREADS+ will be a useful tool to accurately study APA and to analyze gene expression by 3â end counting, especially when the amount of input total RNA is limited. Nine 3'READS+ libraries were made with different amounts (100 ng, 200 ng, 400 ng, 1000 ng, 5000 ng, 15000 ng) of input Hela RNA.

2016-08-14 | E-GEOD-84170 | biostudies-arrayexpress

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