Alternative splicing regulates the physiological adaptation of the mouse hind limb postural and phasic muscles to microgravity

We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both differential gene expression (DGE) and changes in alternative splicing (AS) due to extended spaceflight. Total RNA was isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with translation/ribosomal function and lipid metabolism. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

本研究旨在通过评估长期太空飞行引发的差异基因表达（differential gene expression, DGE）与可变剪接（alternative splicing, AS）变化，全面阐明微重力（microgravity）诱导的小鼠肌肉表型的转录组学基础。本研究从暴露于微重力或地面对照环境达9周的32周龄雌性BALB/c小鼠的后肢姿势性肌腓肠肌（gastrocnemius）与时相性肌股四头肌（quadriceps）中分别提取总RNA。RNA测序（RNA sequencing）结果显示，差异基因表达与可变剪接的变化在姿势性肌与时相性肌中存在差异：腓肠肌中差异基因表达占主导，而股四头肌中可变剪接更为显著。基因本体（gene ontology, GO）分析表明，差异基因表达与可变剪接调控的分子通路各不相同。研究发现，多种编码肌肉骨骼蛋白（Tnnt3、Tnnt1、Neb、Ryr1及Ttn）以及肌肉特异性RNA结合剪接调控因子（Mbnl1、Rbfox1）的非差异表达转录本，其可变剪接发生显著变化，进而改变了其编码蛋白的关键功能结构域。与之形成鲜明对比的是，微重力诱导的差异表达基因与翻译/核糖体功能及脂质代谢密切相关。本研究是首个针对太空飞行过程中大型肢体肌肉内差异基因表达与可变剪接协同变化的全面研究。我们提出，可变剪接对前体mRNA的大规模重塑是骨骼肌适应微重力的转录组学调控的核心环节。本研究鉴定出的可变剪接基因，可通过小分子剪接调控因子疗法进行靶向干预，以缓解太空飞行期间微重力诱导的肌肉变化。