Nuclear 2′-O-methylation regulates RNA splicing through its binding protein FUBP1 (CLIP-seq and PAR-CLIP)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1251610 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR33158755_1.fastq.gz | Fastqsanger.gz | |||
| SRR33158755_2.fastq.gz | Fastqsanger.gz | |||
| SRR33158756_1.fastq.gz | Fastqsanger.gz | |||
| SRR33158756_2.fastq.gz | Fastqsanger.gz | |||
| SRR33158757.fastq.gz | Fastqsanger.gz |
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Project description:2′-O-methylation (Nm) is an abundant RNA modification exists on different mammalian RNA species. However, potential Nm recognition by proteins has not been extensively explored. Here, we employed RNA affinity purification followed by mass spectrometry to identify Nm-binding proteins. The candidates exhibit enriched binding at known Nm sites. Interestingly, some candidates display nuclear localization and functions. We focused on the splicing factor FUBP1. Electrophoretic mobility shift assay (EMSA) validated preference of FUBP1 to Nm-modified RNA. As FUBP1 predominantly binds intronic regions, we profiled Nm sites in chromatin-associated RNA (caRNA) and found Nm enrichment within introns. Depletion of Nm led to increased exon skipping, suggesting Nm-dependent splicing regulation. The caRNA Nm sites overlap with FUBP1 binding sites, and Nm depletion reduced FUBP1 occupancy on modified regions. Furthermore, FUBP1 depletion induced exon skipping in Nm-modified genes, supporting its role in mediating Nm-dependent splicing regulation. Overall, our findings identify FUBP1 as an Nm-binding protein and uncover previously unrecognized nuclear functions for RNA Nm modification.
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Project description:2′-O-methylation (Nm) is an abundant RNA modification exists on different mammalian RNA species. However, potential Nm recognition by proteins has not been extensively explored. Here, we employed RNA affinity purification followed by mass spectrometry to identify Nm-binding proteins. The candidates exhibit enriched binding at known Nm sites. Interestingly, some candidates display nuclear localization and functions. We focused on the splicing factor FUBP1. Electrophoretic mobility shift assay (EMSA) validated preference of FUBP1 to Nm-modified RNA. As FUBP1 predominantly binds intronic regions, we profiled Nm sites in chromatin-associated RNA (caRNA) and found Nm enrichment within introns. Depletion of Nm led to increased exon skipping, suggesting Nm-dependent splicing regulation. The caRNA Nm sites overlap with FUBP1 binding sites, and Nm depletion reduced FUBP1 occupancy on modified regions. Furthermore, FUBP1 depletion induced exon skipping in Nm-modified genes, supporting its role in mediating Nm-dependent splicing regulation. Overall, our findings identify FUBP1 as an Nm-binding protein and uncover previously unrecognized nuclear functions for RNA Nm modification.
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Project description:2′-O-methylation (Nm) is an abundant RNA modification exists on different mammalian RNA species. However, potential Nm recognition by proteins has not been extensively explored. Here, we employed RNA affinity purification followed by mass spectrometry to identify Nm-binding proteins. The candidates exhibit enriched binding at known Nm sites. Interestingly, some candidates display nuclear localization and functions. We focused on the splicing factor FUBP1. Electrophoretic mobility shift assay (EMSA) validated preference of FUBP1 to Nm-modified RNA. As FUBP1 predominantly binds intronic regions, we profiled Nm sites in chromatin-associated RNA (caRNA) and found Nm enrichment within introns. Depletion of Nm led to increased exon skipping, suggesting Nm-dependent splicing regulation. The caRNA Nm sites overlap with FUBP1 binding sites, and Nm depletion reduced FUBP1 occupancy on modified regions. Furthermore, FUBP1 depletion induced exon skipping in Nm-modified genes, supporting its role in mediating Nm-dependent splicing regulation. Overall, our findings identify FUBP1 as an Nm-binding protein and uncover previously unrecognized nuclear functions for RNA Nm modification.
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Project description:Splicing is a central process in metazoans and greatly expands their proteome by alternative splicing of pre-mRNA transcripts. An essential regulatory step during early spliceosome assembly is the recognition of cis-regulatory RNA motifs in pre-mRNAs. Here, we identified the RNA binding protein FUBP1 as a novel core splicing factor with a ubiquitous footprint on pre-mRNAs. FUBP1 binds to a previously unknown cis-regulatory motif upstream of the branch point of human introns. We show that FUBP1 binds and stabilises known 3' splice site components such as the essential splicing factor U2AF2. FUBP1 mutant cell lines and patient data indicate that FUBP1 is particularly relevant for efficient splicing of exons flanked by long introns. In addition to its role at the 3’ splice site, FUBP1 shows multiple interactions with U1 snRNP- associated proteins. This demonstrates an important role for FUBP1 in splice site bridging in the context of long introns.
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Project description:Splicing is a central process in metazoans and greatly expands their proteome by alternative splicing of pre-mRNA transcripts. An essential regulatory step during early spliceosome assembly is the recognition of cis-regulatory RNA motifs in pre-mRNAs. Here, we identified the RNA binding protein FUBP1 as a novel core splicing factor with a ubiquitous footprint on pre-mRNAs. FUBP1 binds to a previously unknown cis-regulatory motif upstream of the branch point of human introns. We show that FUBP1 binds and stabilises known 3' splice site components such as the essential splicing factor U2AF2. FUBP1 mutant cell lines and patient data indicate that FUBP1 is particularly relevant for efficient splicing of exons flanked by long introns. In addition to its role at the 3’ splice site, FUBP1 shows multiple interactions with U1 snRNP- associated proteins. This demonstrates an important role for FUBP1 in splice site bridging in the context of long introns.
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