Rbfox1 controls alternative splicing of focal adhesion genes in cardiac muscle cells

Abstract

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of Vinculin (Metavinculin isoform) and Paxillin (extended Paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell–matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed.

Metavinculin mediates differentiation cues into myogenic gene expression

“…Moreover, Rbfox1 regulates metavinculin expression over Rbfox2 as shown by depletion experiments (Figure 2E-2G) which is strengthened by a recent report. 94 Further, crosslink-RIP analysis confirmed the differentiation-specific switching of Rbfox1 from 5'-to 3'-metavinculin flanking intron (Figure 2H-2J). While ectopic Rbfox1 expression showed induction of metavinculin transcripts under growth-promoting conditions however it is not reflected at protein level which suggested the unstable metavinculin protein in proliferating cells (Figure 1B, 1D and 2K-2N).…”