Abstract
RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR; we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting; MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.
Highlights
Pre-mRNA splicing or alternative splicing is one of the most important mechanisms in post-transcriptional regulation
In the Z-band, no changes were observed in exons 7–10 between wild type (WT) and the RNA binding motif 20 (RBM20) knockout (KO) at different ages, and only one variant (v1) was present in both WT and KO heart tissue (Figure 1A)
While it is known that RBM20 is a splicing factor, it cannot be assumed that all changes in splicing patterns seen in RBM20 KOs result from loss of RBM20 activity at the exons being examined, and loss of RBM20 could have indirect effects on splice variant stability or non-regulated loss of splicing fidelity
Summary
Pre-mRNA splicing or alternative splicing is one of the most important mechanisms in post-transcriptional regulation. Pre-mRNA splicing has been extensively studied and splicing mechanisms of some genes have been well characterized, the splicing mechanisms of numerous genes are still poorly understood given the complexity of splicing regulation of each individual gene. It consists of 363 exons encoding the largest protein known to-date. Studies have shown that titin undergoes numerous splicing events producing millions of isoforms of differing sizes [6,7,8]. The mechanism governing titin splicing had not been deciphered until a new splicing factor, RNA binding motif 20 (RBM20) was identified [9]
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