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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 29, 2016 |
| Title |
The RNA-binding landscape of RBM10 and its role in alternative splicing regulation in models of mouse early development |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Mutations in the RNA-binding protein, RBM10, result in a human syndromic form of cleft palate, termed TARP syndrome. A role for RBM10 in alternative splicing regulation has been previously demonstrated in human cell lines. To uncover the cellular functions of RBM10 in a cell line that is relevant to the phenotype observed in TARP syndrome, we used iCLIP to identify its endogenous RNA targets in a mouse embryonic mandibular cell line. We observed that RBM10 binds to pre-mRNAs with significant enrichment in intronic regions, in agreement with a role for this protein in pre-mRNA splicing. In addition to protein-coding transcripts, RBM10 also binds to a variety of cellular RNAs, including non-coding RNAs, such as spliceosomal small nuclear RNAs, U2 and U12. RNA-seq was used to investigate changes in gene expression and alternative splicing in RBM10 KO mouse mandibular cells and also in mouse ES cells. We uncovered a role for RBM10 in the regulation of alternative splicing of common transcripts in both cell lines but also identified cell-type specific events. Importantly, those pre-mRNAs that display changes in alternative splicing also contain RBM10 iCLIP tags, suggesting a direct role of RBM10 in these events. Finally, we show that depletion of RBM10 in mouse ES cells leads to proliferation defects and to gross alterations in their differentiation potential. These results demonstrate a role for RBM10 in the regulation of alternative splicing in two cell models of mouse early development and suggests that mutations in RBM10 could lead to splicing changes that affect normal palate development and cause human disease.
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| Overall design |
We carried out the iCLIP-seq of endogenous mouse RBM10 protein in 4 independent replicates in a mouse mandibular MEPA (Mouse Embryonic Pharyngeal Arch) cell line. For each replicate, we included a control iCLIP corresponding to an IP with rabbit IgG. Reads were mapped to the mouse genome (mm9) and we deduced the crosslink nucleotide counts for each genomic position.
We performed RNA-Seq on polyA+ RNA isolated from RBM10 Knockout in md MEPA and ES cells.
RBM10 KO md MEPA cells were obtained using CRIPR/Cas9 approach. We extracted RNAs from 4 independent RBM10 KO md cell lines (KO1 and KO2 with deletion in exon 3 and KO3 and KO4 with deletion in exon 9) and from WT md MEPA cells (Cont) in triplicates.
RBM10 KO ES cell line, that correspond to the male gene trap cell line Rbm10Gt(CSI176)Byg, was obtained from Baygenomics. We also obtained the parental cell line E14Tg2a.4 from Baygenomics. RNAs were extracted from RBM10 KO and Cont ES cells in triplicates.
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| Contributor(s) |
Rodor J, FitzPatrick DR, Eyras E, Cáceres JF |
| Citation(s) |
27763814 |
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| Submission date |
Oct 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Julie Rodor |
| Organization name |
University of Edinburgh
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| Department |
MRC Human Genetics Unit
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| Lab |
Caceres group
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| Street address |
Western General Hospital
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| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA351215 |
| SRA |
SRP092246 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE89270_RAW.tar |
38.5 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE89270_RNAseq_RBM10_KO_ES_transcript_tpm.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSE89270_RNAseq_RBM10_KO_md_transcript_tpm.txt.gz |
1.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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