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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE32440 | GEO | 2013/02/25

SECONDARY ACCESSION(S): PRJNA147713

REPOSITORIES: GEO

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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 3M-bM-^@M-2 untranslated regions (3M-bM-^@M-^YUTRs), which recruit regulatory proteins and RNAs. More than half of the mammalian genes generate multiple mRNA isoforms differing in their 3M-bM-^@M-2UTRs and therefore in their regulatory elements. The Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1) binds to cognate sites in 3M-bM-^@M-^Y 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 3M-bM-^@M-^Y 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 3M-bM-^@M-^Y splice site recognition by U2AF. RNA-Seq experiments reveal that CPEB1 mediates 3M-bM-^@M-^Y UTR shortening of hundreds of mRNAs, leading to changes in their translation efficiency. Three total RNA from three biological replicates were labeled and hybridized versus its own control in direct and dye-swap hybridizations.

2013-02-25 | E-GEOD-32440 | biostudies-arrayexpress

Mechanisms for U2AF to define 3M-bM-^@M-2 splice sites and regulate alternative splicing in the human genome

Project description:The U2AF heterodimer has been well studied for its role in defining functional 3M-bM-^@M-^Y splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3M-bM-^@M-^Y splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3M-bM-^@M-^Y splice site associated with either the alternative exon to cause exon skipping or with the competing constitutive exon to induce exon inclusion. We further demonstrate partial functional impairment with mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states. Examination of U2AF heterodimer regulated splicing in Hela cells with CLIP-seq (U2AF65), paired-end RNA-seq (si-NC and si-U2AF65) and RASL-seq (respective three biological replicates of WT, si-NC, si-U2AF65, si-U2AF35, si-NC + pcDNA3.0, si-U2AF65 + pcDNA3.0, and si-U2AF65 + Flag-U2AF35)

2014-10-08 | E-GEOD-61603 | biostudies-arrayexpress

Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome

Project description:The U2AF heterodimer has been well studied for its role in defining functional 3’ splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3’ splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3’ splice site associated with either the alternative exon to cause exon skipping or with the competing constitutive exon to induce exon inclusion. We further demonstrate partial functional impairment with mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.

2014-10-08 | GSE61603 | GEO

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

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

Project description:Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects. HeLa cell line was stably transfected with shRNA plasmids targeting CstF64. Total RNA was isolated from CstF64 KD cells and wild-type control cells using Trizol according to manufacturerâs protocols. Samples were deep sequenced in duplicate using the Illumina GAIIx system.

2016-03-14 | E-GEOD-79157 | biostudies-arrayexpress

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

2016-03-14 | GSE79157 | 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

RNA binding protein CPEB1 remodels host and viral RNA landscapes [RNA-Seq]

Project description:In this study, we report that HCMV infection results in widespread alternative splicing (AS), shorter 3′-untranslated regions (3′UTRs) and polyA tail lengthening in host genes and CPEB1 depletion reverses infection-related post-transcriptional changes.

2016-09-17 | GSE86791 | GEO

Exon array analysis of resting and activated primary murine CD4+ T-cells

Project description:The stability, localization and translation of mammalian mRNAs are largely determined by sequences in the 3' untranslated region (UTR). Here, we describe a conserved program of increased upstream polyadenylation site usage following activation of primary murine CD4+ T lymphocytes. This program, resulting in shorter 3' UTRs, is a characteristic of immune cell activation and strongly correlates with proliferation across diverse cell types and tissues. Enforced expression of full-length 3' UTRs confers differences in protein expression that can in some cases be eliminated by deletion of predicted microRNA target sites in the variably included region. Together, our data indicate that polyadenylation site usage is coordinately regulated such that states of increased proliferation are associated with widespread reduction of 3' UTR-based regulation. Keywords: activation, exon array, time course Two or three biological replicates per time point

2010-05-26 | E-GEOD-10666 | biostudies-arrayexpress

UHMK1 is a novel splicing regulatory kinase

Project description:The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif (UHM), a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3’ splice site recognition and early steps of spliceosome assembly. Also, UHMK1 phosphorylates these splicing factors in vitro. However, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we used global phosphoproteomics to identify novel putative substrates in UHMK1-overexpressing or -depleted (knockdown) NIH3T3 cells and better characterize the function of this kinase in splicing and other biological processes.

2023-05-02 | PXD033353 | Pride

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