Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE79792 | GEO | 2017/08/10
SECONDARY ACCESSION(S): PRJNA317002
REPOSITORIES: GEO
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Project description:RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA-protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins-SRSF3 and SRSF7, regulators of pre-mRNA splicing, nuclear export and translation-interact with RNA in different cellular compartments. To do so, we developed Fractionation iCLIP (Fr-iCLIP), in which chromatin, nucleoplasmic and cytoplasmic fractions are prepared from UV-crosslinked cells and then subjected to iCLIP. As expected, SRSF3 and SRSF7 targets were detected in all fractions, with intron, snoRNA and lncRNA interactions enriched in the nucleus. Cytoplasmically-bound mRNAs reflected distinct functional groupings, suggesting coordinated translation regulation. Surprisingly, hundreds of cytoplasmic intron targets were detected. These cytoplasmic introns were found to be highly conserved and introduced premature termination codons into coding regions. However, many intron-retained mRNAs were not substrates for nonsense-mediated decay (NMD), even though they were detected in polysomes. These findings suggest that intron-retained mRNAs in the cytoplasm have previously uncharacterized functions and/or escape surveillance. Hence, Fr-iCLIP detects the cellular location of RNA-protein interactions and provides insight into co-transcriptional, post-transcriptional and cytoplasmic RBP functions for coding and non-coding RNAs.
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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 | GSE151720 | GEO
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.
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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.
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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.
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2016-02-17 | E-GEOD-71095 | biostudies-arrayexpress