Molecular Analysis of Alternative Spliced Transcripts of Equine Pleckstrin Homology and RhoGEF Domain Containing G1 (PLEKHG1) gene

Abstract

Since horse industry get bigger with horse racing and horse riding, athletic performance become most important trait on horses, but the molecular analysis and regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we conducted RNA-sequencing analysis with muscle and blood samples by exercise in Thoroughbreds. Through RNA-sequencing, we identified Pleckstrin Homology and RhoGEF Domain Containing G1 (PLEKHG1) gene expressed differentially by alternative spliced isoforms in skeletal muscle during exercise. In this study, we conducted reverse transcriptase polymerase chain reaction (RT-PCR) to validate two isoforms of equine PLEKHG1 transcripts (PLEKHG1a, PLEKHG1b), and cloned the transcripts to confirm the sequences. Additionally, we validated expression pattern of PLEKHG1a (long form of transcript) and PLEKHG1b (short form of transcript) in horse tissue by quantitative RT-PCR (qRT-PCR). Prediction of protein structure of these isoforms revealed two putative phosphorylation sites at the amino acid sequences encoded in exon 7, which is deleted in PLEKHG1b. Expression pattern of PLEKHG1a and PLEKHG1b shows cross expressed pattern as RNA-sequencing data. PLEKHG1a increased after exercise whereas PLEKHG1b decreased after exercise. Collectively, it is assumed that the expression patterns of PLEKHG1a and PLEKHG1b transcripts would be involved in regulation of myogenic differentiation and myogenic proliferation through the Ras signaling pathway. Further study should be necessary to uncover biological function(s) and significance of the alternative splicing isoforms in equine skeletal muscle