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. Overall design: 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.

可变RNA剪接（Alternative RNA Splicing，AS）是一种高度保守的转录后调控机制，可通过生成mRNA变体以拓展蛋白质组多样性。急性耐力运动似乎会短暂扰动骨骼肌中的可变剪接事件，但目前学界对其全转录组层面的应答机制尚未完全阐明。本研究旨在通过对比短读长RNA测序（Short-read RNA Sequencing，SRS）与长读长RNA测序（Long-read RNA Sequencing，LRS）数据，更好地解析骨骼肌中的差异可变剪接（Differential Alternative RNA Splicing，DAS）。

本研究从基因表达综合数据库（Gene Expression Omnibus，GEO）中提取了公开可用的临床运动研究相关短读长RNA测序数据，并对跑步机运动前后的小鼠腓肠肌开展了牛津纳米孔（Oxford Nanopore）长读长RNA测序。研究分析了差异基因表达（Differential Gene Expression，DGE）、差异可变剪接（DAS）以及异构体转换事件，并通过蛋白质印迹（Western Blot）实验验证了候选基因的表达变化。

短读长与长读长测序均显示，运动后骨骼肌中存在显著的差异基因表达现象，分别有57种和15种RNA结合蛋白（RNA-binding Proteins，RBPs）出现上调与下调。对短读长测序数据的rMATS分析显示，外显子跳跃与内含子保留是最为常见的剪接事件类型。对长读长测序数据的Swan分析显示，共有38种RNA结合蛋白存在显著的异构体转换现象，其中Hnrnpa3发生了从内含子保留型到蛋白编码型的显著转换。蛋白质印迹验证显示，运动后1小时（p=0.0043）与24小时（p=0.0103），HnRNP-A3蛋白水平均出现近两倍的上调。

本研究表明，急性耐力运动可引发骨骼肌中与可变剪接相关的应答变化以及RNA结合蛋白的表达改变。短读长RNA测序虽是分析差异基因表达的有力工具，但无法有效检测可变剪接事件。因此，那些仅存在差异可变剪接与蛋白表达变化但无转录水平改变的“隐藏”基因，仍是当前研究的主要知识空白。本研究证实，长读长RNA测序可揭示此前未被发现的转录本多样性以及调控可变剪接的分子机制。

实验设计概述：为探究急性耐力运动对骨骼肌可变剪接的影响，本研究选取20只小鼠进行急性跑步机运动：以最大速度的60%恒定配速持续运动30分钟。随后在四个时间点处死小鼠，采集其腓肠肌组织，经液氮快速冷冻后保存于-80℃冰箱，用于后续组织处理与RNA提取。本研究随后分离含poly(A)尾的RNA，构建带条形码的PCR-cDNA文库（牛津纳米孔科技公司，Oxford Nanopore Technologies），并使用搭载MinION™ R9.4.1流动槽的MinION™ Mk1C测序仪开展长读长RNA测序。