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Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

ABSTRACT: The precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3’UTR. We previously reported the important diversity of 3’UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.

ORGANISM(S): Mus musculus

PROVIDER: GSE183704 | GEO | 2021/09/10

REPOSITORIES: GEO

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A jack of all trades and a master of some: remodeling of signaling pathways downstream of CFIm 3’ end processing factor

Project description:The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To uncover the molecular pathways that translate CFIm expression levels into these observed phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that more than 600 genes exhibit CFIm-dependent APA, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the metabolic and proliferation changes induced by CFIm perturbations. Transcripts of miRNA pathway proteins are prominent targets of CFIm-dependent APA. This leads to increased miRNA biogenesis and repressive activity at the same time as 3’ UTRs become shorter upon CFIm depletion. Our study provides a first systematic assessment of the signalling pathways downstream of CFIm to improve our understanding of CFIm-dependent phenotypes.

2022-02-11 | PXD027034 | Pride

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis

Project description:The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that more than 800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3’ UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a reference set of core APA targets, that respond to changes in CFIm protein subunit levels (CFIm25/CFIm68), and the elicited signalling pathways downstream of CFIm, to improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

2022-02-16 | GSE179630 | GEO

A unified activation mechanism for mRNA 3’ end processing and splicing mediated by SR superfamily proteins

Project description:Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3’ processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3’ processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PAS). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is modulated by phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like regions in regulatory and core factors may provide a common activation mechanism for mRNA 3’ processing, splicing, and potentially other steps in RNA metabolism.

2017-11-01 | GSE101871 | 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

Identification of the JNK Signaling Pathway as a Functional Target of the Tumor Suppressor PTEN

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2007-06-01 | GSE7562 | GEO

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2014-05-15 | E-GEOD-42420 | biostudies-arrayexpress

RNA-seq of P19 cells after knockdown of Srsf3, Srsf7 and Cpsf6 and after differentiation into neuronal cells

Project description:Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3’ untranslated regions (3’UTRs). APA regulates stage- and tissue-specific gene expression by affecting the stability, subcellular localization and translation rate of transcripts. We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained, however, unknown. Here, we combined iCLIP, RNA-Seq and 3’-end sequencing to find that both proteins bind upstream of proximal PASs (pPASs), yet they exert opposite effects on 3’UTR length. We show that SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, thereby generating short 3’UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, and hypo-phosphorylation contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3’UTRs by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon reduced expression of SRSF3, CFIm levels strongly decrease and 3’UTRs are globally shortened. In SRSF3-regulated transcripts, CFIm and FIP1 bind upstream of dPASs and promote their usage. Surprisingly, both factors are also recruited to pPASs under conditions where their usage is blocked, suggesting the formation of inactive cleavage complexes. Thus, we identify SRSF3 as a novel regulator of CFIm activity, provide evidence that CFIm inhibits pPAS usage and show that small differences in the domain architecture of SR proteins confer opposite effects on PAS selection.

2021-01-28 | GSE151721 | GEO

Transcriptome-wide mapping of RNA:protein interactions of CPSF5 and FIP1 in P19 cells by iCLIP

2021-01-28 | GSE151720 | GEO

MACE-seq of P19 cells after knockdown of Srsf3 and Srsf7

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PTEN loss deM-oM-,M-^Anes a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma

Project description:Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be deM-oM-,M-^Aned by gene expression proM-oM-,M-^Aling. However, even within these deM-oM-,M-^Aned subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/ protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotox- icity in PTEN-deM-oM-,M-^Acient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deM-oM-,M-^Acient GCB DLBCL lines. Collectively, our results indicate that PTEN loss deM-oM-,M-^Anes a PI3K/ AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas. This GEO dataset is comprised of a) GEP measurements for 34 primary DLBCL patient samples plus two reference samples, b) 8 paired GEP measurements of the HT DLBCL cell line and c) aCGH measurements for two DLBCL cell lines in addition to previously published cell lines in GSE43272 (i.e., Sample GSM1059798). All of these data were used in the paper cited below.

2013-07-09 | E-GEOD-45495 | biostudies-arrayexpress

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