Impact of Acute Endurance Exercise on Alternative Splicing in Skeletal Muscle

II: ABSTRACT Purpose Alternative 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) and differential isoform expression (DIE) in skeletal muscle by comparing short-read (SRS) and long-read RNA sequencing (LRS) data. Methods 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 (30m running, n =5 mice/group, 20 total, 10 weeks old). Differential gene expression (DGE) and DIE were analyzed and validated using RT-PCR and immunoblots. Results Both SRS and LRS illustrated significant DGE in skeletal muscle post-exercise, including 89 RNA-binding proteins (RBPs). rMATS analysis of SRS revealed that exon-skipping and intron-retaining events were the most common. Swan analysis of LRS revealed several common genes across post-exercise cohorts with significant DAS but no DGE: 13 exercise-associated genes, including mSirt2 (24.5% shift at 24hr post-exercise [24pe], p =0.005); 61 RBPs, including mHnrnpa3 (28.5% at 24pe, p =0.02), mHnrnpa1 (30.6% at 24pe, p =0.004), and mTia1 (53.6% at 24pe, p =0.004). Conclusions We illustrated that acute endurance exercise can elicit changes in AS-related responses and RBP expression in skeletal muscle, especially at 24pe. SRS is a powerful tool for analyzing DGE but lacks isoform detection, posing a major gap in knowledge of “hidden” genes with no transcriptional but significant DIE and protein expression changes. Additionally, LRS can uncover previously unknown transcript diversity and mechanisms influencing endurance exercise adaptations and responses.