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Identification of RBM5 and RBM10-dependent splicing events in 293Flp-In cells.

ABSTRACT: RBM5 and RBM10 are RNA-binding proteins and splicing regulators. These two proteins are putative paralogs in mammalian cells, sharing common domain organization and extensive protein sequence similarity, but their RNA-binding preferences differ. We developed a sensitive system to identify splicing events regulated by RBM5 and/or RBM10, deleting all RBM5 alleles in 293Flp-In cells, and reducing the expression of RBM10, which is partially rescued by activation of a cryptic exon (CE) in one of the RBM10 alleles. RBM5 or RBM10 transgenes were then introduced in these RBM5-null, RBM10 mutant cells (RBM5-/-;RBM10-/CE), allowing controlled protein expression, induced with Doxycycline. Using this system we identify thousands of RBM5 and RBM10-regulated cassette exons, and note a substantial overlap between the two sets.

ORGANISM(S): Homo sapiens

PROVIDER: GSE240259 | GEO | 2024/03/08

REPOSITORIES: GEO

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Regulation of NUMB alternative splicing by RBM5, RBM6 and RBM10 controls cancer cell proliferation (CLIP-Seq)

Project description:RBM5, a regulator of alternative splicing of apoptotic genes, and its close homologues, RBM6 and RBM10, are RNA binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation and its modulation recapitulates or antagonizes the effects of RBM5, 6 and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation. CLIP-Seq analysis of RBM5, RBM6 and RBM10, with 2 biological replicates and one non-specific control for each protein.

2013-12-20 | E-GEOD-48066 | biostudies-arrayexpress

Regulation of NUMB alternative splicing by RBM5, RBM6 and RBM10 controls cancer cell proliferation

Project description:RBM5, a regulator of alternative splicing of apoptotic genes, and its close homologues, RBM6 and RBM10, are RNA binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation and its modulation recapitulates or antagonizes the effects of RBM5, 6 and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation. RNA from 3 biological replicates of knockdowns of RBM5, 6 and 10 and a control set were used. Changes between the control and knockdowns were measured based on using a splice-junction array (Affymetrix HJAY).

2013-12-20 | E-GEOD-47431 | biostudies-arrayexpress

Regulation of NUMB alternative splicing by RBM5, RBM6 and RBM10 controls cancer cell proliferation

2013-12-20 | GSE47431 | GEO

Regulation of NUMB alternative splicing by RBM5, RBM6 and RBM10 controls cancer cell proliferation (CLIP-Seq)

2013-12-20 | GSE48066 | GEO

Global analysis of alternative splicing regulated by RBM10

Project description:RBM10 is an RNA binding protein that was identified as a component of spliceosome complex, suggesting its potential role in splicing regulation. However, the direct experimental evidence for this function has been lacking. Here we characterized in vivo RBM10-RNA interactions and investigated the role of RBM10 in splicing regulation at the global level. We observed significant RBM10-RNA interactions in the vicinity of splice sites and identified hundreds of splicing changes following perturbation of cellular RBM10 abundance. A RNA splicing map integrating the binding pattern and splicing profiles revealed a significant correlation between RBM10-enhanced exon skipping events and its binding close to the splicing sites of both upstream and downstream introns. Furthermore, we demonstrated the splicing defects in a patient carrying a RBM10 mutation. Overall, our data provided insights into the mechanistic model of RBM10-mediated splicing regulation and established genomic resources for future studies on its function in different pathophysiological contexts. We sequenced the mRNA of HEK293 cells and LCL cells, and we determined the RBM10 binding sites using PARCLIP in HEK293 cells. In total we sequenced four mRNA-Seq libraries for KD and two for OE in HEK293 cells; for each of these libraries, we also sequenced one control library. We also sequenced the mRNA of one patient LCL and two normal LCL libraries. Two replicates of PARCLIP sequencing were perfomed.

2013-09-03 | E-GEOD-44976 | biostudies-arrayexpress

Global analysis of alternative splicing regulated by RBM10

2013-09-03 | GSE44976 | GEO

Rbm10 promotes heterochromatin assembly by interacting with Clr6 and chromatin remodeling complexes

Project description:Splicing factors have recently been shown to be involved in heterochromatin formation, but their role in controlling heterochromatin structure and function remains poorly understood. In this study, we identified a fission yeast homologue of human splicing factor RBM10, which has been linked to TARP syndrome. Overexpression of Rbm10 in fission yeast leads to strong global intron retention. Rbm10 also interacts with splicing factors in a pattern resembling that of human RBM10, suggesting that the function of Rbm10 as a splicing regulator is conserved. Surprisingly, our deep-sequencing data showed that deletion of Rbm10 caused only minor effect on genome-wide gene expression and splicing. However, the mutant displays severe heterochromatin defects. Further analyses confirmed that the heterochromatin defects in the mutant did not result from mis-splicing of heterochromatin factors. Our proteomic data revealed that Rbm10 associates with the histone deacetylase Clr6 complex and chromatin remodeling complexes known to be essential for heterochromatin silencing. We propose that Rbm10 serves as a platform to recruit Clr6 and chromatin remodelers to mediate heterochromatin formation. Our work together with previous findings further suggests that different splicing subunits may play distinct roles in heterochromatin regulation.

2017-01-23 | GSE93906 | GEO

The Effect of RBM5 Gene Manipulation on Expression Data from Day In Vitro 6 Primary Rat Cortical Neurons

Project description:RNA Binding Motif 5 (RBM5) is a nuclear splicing factor. Prior studies show that RBM5 overexpression in cancer cells alters gene splicing, gene expression, and induces cell death and/or growth arrest. The role of RBM5 in primary neurons is unknown, and its potential mRNA targets have not been identified. Using lentivirus based approaches we tested if RBM5 knockdown (i.e. by shRNA) or RBM5 overexpression (DDK-tagged rat open reading frame) alters whole genome expression in primary rat cortical neurons. We used microarrays to examine genes that are up-regulated vs. down-regulation in cortical neurons after RBM5 knockdown vs. overexpression.

2018-02-16 | GSE98858 | GEO

RNP-seq and IP-seq from cells expressing epitope-tagged RBM5, RBM10, or SF3A3

Project description:We isolated snRNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. These complexes contained U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. The complexes have similar composition, whether directly isolated from extracted material, or after selection from glycerol gradients. Here we describe sequencing libraries prepared from RNA derived from directly isolated (IP-seq), or gradient-purified complexes (RNP-seq). Libraries from deproteinized RNA were prepared after selective degradation of U2 snRNA, each in a triplicate. An additional library from RBM5-Flag RNP complex was prepared without such degradation. The majority of sequenced pre-mRNA fragments map to intronic branch sites.

2024-03-08 | GSE240596 | GEO

Inactivation of the splicing factor RBM10 limits EGFR targeted therapy response in EGFR mutant lung cancer

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2022-06-04 | GSE199240 | GEO

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