Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA

Abstract

BackgroundRBM10 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.ResultsRBM10 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.ConclusionOur work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-017-0096-x) contains supplementary material, which is available to authorized users.

Mutational landscape of non-muscle-invasive bladder cancer

“…Inactivating mutations appear to span the entire grade/stage spectrum of NMIBC ( Table 1). Knockdown of RBM10 leads to altered splicing of a wide range of genes in cancer cell lines, several of which are associated with RAS signaling [50].…”

RBM10: Harmful or helpful‐many factors to consider

“…RBM10 mutation has particular relevance to TARP syndrome, and may have relevance to spinal muscular atrophy (SMA) . Unfortunately, actual changes in RBM10 expression have not been rigorously examined in either of these diseases.…”

A gene mutation in RNA‑binding protein 10 is associated with lung adenocarcinoma progression and poor prognosis

“…Moreover, alteration of mRNA alternative splicing, especially exon skipping ( 7 ) or exon-inclusion of the affected proteins ( 7 , 9 ), results in abnormal protein expression and functions in cells. RBM10 knockdown provoked alterations in 10–20% of the pre-mRNA splicing events ( 10 ), while other studies showed that RBM10 is a tumor suppressor gene ( 11 , 12 ) Thus, RBM10 may play an important role in suppression of human tumorigenesis. Previous studies showed that RBM10 mutations did occur in lung and pancreatic cancers ( 13 , 14 ) and the Cancer Genome Atlas (TCGA) data showed that RBM10 mutations occurred in 8% of lung adenocarcinoma and is more common in male patients.…”