Impact of Acute Exercise on Alternative Splicing in Skeletal Muscle

Alternative RNA splicing (AS) is a highly conserved post-transcriptional mechanism, generating mRNA variants to diversify the proteome. Acute endurance exercise appears to transiently perturb AS in skeletal muscle, but transcriptome-wide responses are not well-defined. We aimed to better understand differential AS (DAS) in skeletal muscle by comparing short-read RNA sequencing (SRS) and long-read RNA sequencing (LRS) data. Publicly accessible SRS of clinical exercise studies were extracted from the Gene Expression Omnibus. Oxford Nanopore LRS was performed on mouse gastrocnemius before and following treadmill exercise. Differential gene expression (DGE), DAS, and isoform switching were analyzed. Western blots were performed to validate expression changes of candidate genes. Both SRS and LRS illustrated significant DGE in skeletal muscle post-exercise, whereby 57 and 15 RNA-binding proteins (RBPs) were up-/down-regulated, respectively. rMATS analysis of SRS data revealed that exon-skipping and intron-retaining splicing events were the most common. Swan analysis of LRS data revealed 38 RBPs with significant isoform switching: one of these RBPs, Hnrnpa3, underwent a significant intron-retained to protein-coding switch. HnRNP-A3 protein levels validated nearly two-fold increases at 1 hour (p=.0043) and 24 hours (p=.0103) post-exercise. This study illustrates that acute endurance exercise can elicit changes in AS-related responses and RBP expression in skeletal muscle. SRS is certainly a powerful tool for analyzing DGE but lacks AS detection. As such, “hidden” genes with no transcriptional changes but significant DAS and protein expression changes pose a major gap in knowledge. Our work highlights how LRS can uncover previously unknown transcript diversity and mechanisms influencing AS. To investigate the impact of acute endurance exercise on alternative splicing in skeletal muscle, 20 mice underwent acute treadmill running (60% of maximal speed) treadmill running at a constant pace for 30 minutes. The mice were sacrificed at four time points, whereby the gastrocnemius muscles were harvested, flash frozen in liquid nitrogen, and stored at –80°C for subsequent tissue processing and RNA isolation. We then isolated poly(A)-containing RNA to synthesize barcoded PCR-cDNA libraries (Oxford Nanopore Technologies) for long-read RNA sequencing on the MinION™ Mk1C sequencer with a MinION™ R9.4.1 flow cell.