Smooth muscle has a central role in bronchospasm-induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis-regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT-PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis-acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.
Keywords: Airway smooth muscle; SMB isoform; SRSF1; SRSF6; alternative splicing; asthma; hnRNPA1.
© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.