Co- and post-transcriptional regulation of Rbm5 and Rbm10 in mouse cells as evidenced by tissue-specific, developmental and disease-associated variation of splice variant and protein expression levels.

BACKGROUND: Expression and function of the two RNA binding proteins and regulators of alternative splicing, RBM5 and RBM10, have largely been studied in human tissue and cell lines. The objective of the study described herein was to examine their expression in mouse tissue, in order to lay the framework for comprehensive functional studies using mouse models. METHODS: All RNA variants of Rbm5 and Rbm10 were examined in a range of normal primary mouse tissues. RNA and protein were examined in differentiating C2C12 myoblasts and in denervated and dystonin-deficient mouse skeletal muscle. RESULTS: All Rbm5 and Rbm10 variants examined were expressed in all mouse tissues and cell lines. In general, Rbm5 and Rbm10 RNA expression was higher in brain than in skin. RNA expression levels were more varied between cardiac and skeletal muscle, depending on the splice variant: for instance, Rbm10v1 RNA was higher in skeletal than cardiac muscle, whereas Rbm10v3 RNA was higher in cardiac than skeletal muscle. In mouse brain, cardiac and skeletal muscle, RNA encoding an approximately 17kDa potential paralogue of a small human RBM10 isoform was detected, and the protein observed in myoblasts and myotubes. Expression of Rbm5 and Rbm10 RNA remained constant during C2C12 myogenesis, but protein levels significantly decreased. In two muscle disease models, neither Rbm10 nor Rbm5 showed significant transcriptional changes, although significant specific alternative splicing changes of Rbm5 pre-mRNA were observed. Increased RBM10 protein levels were observed following denervation. CONCLUSIONS: The varied co-transcriptional and post-transcriptional regulation aspects of Rbm5 and Rbm10 expression associated with mouse tissues, myogenesis and muscle disease states suggest that a mouse model would be an interesting and useful model in which to study comprehensive functional aspects of RBM5 and RBM10.

Insight into the role of alternative splicing within the RBM10v1 exon 10 tandem donor site.

BACKGROUND: RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. Mutations in RBM10, which maps to the X chromosome, are associated with TARP syndrome, lung and pancreatic cancers. Two predominant isoforms of RBM10 exist, RBM10v1 and RBM10v2. Both variants have alternate isoforms that differ by one valine residue, at amino acid 354 (RBM10v1) or 277 (RBM10v2). It was recently observed that a novel point mutation at amino acid 354 of RBM10v1, replacing valine with glutamic acid, correlated with preferential expression of an exon 11 inclusion variant of the proliferation regulatory protein NUMB, which is upregulated in lung cancer. FINDINGS: We demonstrate, using the GLC20 male-derived small cell lung cancer cell line - confirmed to have only one X chromosome - that the two (+/-) valine isoforms of RBM10v1 and RBM10v2 result from alternative splicing. Protein modeling of the RNA Recognition Motif (RRM) within which the alteration occurs, shows that the presence of valine inhibits the formation of one of the two α-helices associated with RRM tertiary structure, whereas the absence of valine supports the α-helical configuration. We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform. This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant. CONCLUSIONS: Our observations suggest that the ability of RBM10v1 to regulate alternative splicing depends, at least in part, on a structural alteration within the second RRM domain, which influences whether RBM10v1 functions to support or repress splicing. A model is presented.