PMID- 28728573 OWN - NLM STAT- MEDLINE DCOM- 20180423 LR - 20181113 IS - 1471-2199 (Electronic) IS - 1471-2199 (Linking) VI - 18 IP - 1 DP - 2017 Jul 20 TI - Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA. PG - 19 LID - 10.1186/s12867-017-0096-x [doi] LID - 19 AB - BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion. FAU - Sutherland, Leslie C AU - Sutherland LC AUID- ORCID: 0000-0002-8619-1834 AD - Health Sciences North Research Institute, Sudbury, ON, P3E 5J1, Canada. lsutherland@hsnri.ca. AD - Biomolecular Sciences Program, Laurentian University, Sudbury, ON, P3E 2C6, Canada. lsutherland@hsnri.ca. AD - Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, P3E 2C6, Canada. lsutherland@hsnri.ca. FAU - Thibault, Philippe AU - Thibault P AD - RNomics Platform of Universite de Sherbrooke, Sherbrooke, QC, Canada. FAU - Durand, Mathieu AU - Durand M AD - RNomics Platform of Universite de Sherbrooke, Sherbrooke, QC, Canada. FAU - Lapointe, Elvy AU - Lapointe E AD - RNomics Platform of Universite de Sherbrooke, Sherbrooke, QC, Canada. FAU - Knee, Jose M AU - Knee JM AD - Health Sciences North Research Institute, Sudbury, ON, P3E 5J1, Canada. FAU - Beauvais, Ariane AU - Beauvais A AD - Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada. FAU - Kalatskaya, Irina AU - Kalatskaya I AD - Ontario Institute for Cancer Research, MaRS Centre, Toronto, ON, M5G 0A3, Canada. FAU - Hunt, Sarah C AU - Hunt SC AD - Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, P3E 2C6, Canada. FAU - Loiselle, Julie J AU - Loiselle JJ AD - Biomolecular Sciences Program, Laurentian University, Sudbury, ON, P3E 2C6, Canada. FAU - Roy, Justin G AU - Roy JG AD - Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, P3E 2C6, Canada. FAU - Tessier, Sarah J AU - Tessier SJ AD - Health Sciences North Research Institute, Sudbury, ON, P3E 5J1, Canada. FAU - Ybazeta, Gustavo AU - Ybazeta G AD - Health Sciences North Research Institute, Sudbury, ON, P3E 5J1, Canada. FAU - Stein, Lincoln AU - Stein L AD - Ontario Institute for Cancer Research, MaRS Centre, Toronto, ON, M5G 0A3, Canada. FAU - Kothary, Rashmi AU - Kothary R AD - Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada. AD - Departments of Medicine and of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, K1H 8M5, Canada. FAU - Klinck, Roscoe AU - Klinck R AD - RNomics Platform of Universite de Sherbrooke, Sherbrooke, QC, Canada. AD - Departement de Microbiologie et d'infectiologie, Faculte de Medecine et des Sciences de la Sante, Universite de Sherbrooke, Sherbrooke, QC, Canada. FAU - Chabot, Benoit AU - Chabot B AD - Departement de Microbiologie et d'infectiologie, Faculte de Medecine et des Sciences de la Sante, Universite de Sherbrooke, Sherbrooke, QC, Canada. LA - eng GR - CIHR/Canada PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20170720 PL - England TA - BMC Mol Biol JT - BMC molecular biology JID - 100966983 RN - 0 (RBM10 protein, human) RN - 0 (RNA Precursors) RN - 0 (RNA-Binding Proteins) RN - 0 (SMN2 protein, human) RN - 0 (Survival of Motor Neuron 2 Protein) RN - EC 3.6.5.2 (ras Proteins) SB - IM MH - Alternative Splicing MH - Cell Line MH - Cluster Analysis MH - Computational Biology/methods MH - Exons MH - Fibroblasts MH - Gene Expression Profiling MH - Humans MH - RNA Precursors/*genetics MH - *RNA Splicing MH - RNA-Binding Proteins/*metabolism MH - Reproducibility of Results MH - Signal Transduction MH - Survival of Motor Neuron 2 Protein/genetics MH - ras Proteins/metabolism PMC - PMC5520337 OTO - NOTNLM OT - Alternative splicing OT - Cancer OT - RBM10 OT - SMN2 OT - Spinal muscular atrophy OT - Splicing array EDAT- 2017/07/22 06:00 MHDA- 2018/04/24 06:00 PMCR- 2017/07/20 CRDT- 2017/07/22 06:00 PHST- 2017/05/15 00:00 [received] PHST- 2017/07/14 00:00 [accepted] PHST- 2017/07/22 06:00 [entrez] PHST- 2017/07/22 06:00 [pubmed] PHST- 2018/04/24 06:00 [medline] PHST- 2017/07/20 00:00 [pmc-release] AID - 10.1186/s12867-017-0096-x [pii] AID - 96 [pii] AID - 10.1186/s12867-017-0096-x [doi] PST - epublish SO - BMC Mol Biol. 2017 Jul 20;18(1):19. doi: 10.1186/s12867-017-0096-x.