Alternative splicing diversifies the skeletal muscle transcriptome during prolonged spaceflight.

As the interest in manned spaceflight increases, so does the requirement to understand the transcriptomic mechanisms that underlay the detrimental physiological adaptations of skeletal muscle to microgravity. While microgravity-induced differential gene expression (DGE) has been extensively investigated, the contribution of differential alternative splicing (DAS) to the plasticity and functional status of the skeletal muscle transcriptome has not been studied in an animal model. Therefore, by evaluating both DGE and DAS across spaceflight, we set out to provide the first comprehensive characterization of the transcriptomic landscape of skeletal muscle during exposure to microgravity. RNA-sequencing, immunohistochemistry, and morphological analyses were conducted utilizing total RNA and tissue sections isolated from the gastrocnemius and quadriceps muscles of 30-week-old female BALB/c mice exposed to microgravity or ground control conditions for 9 weeks. In response to microgravity, the skeletal muscle transcriptome was remodeled via both DGE and DAS. Importantly, while DGE showed variable gene network enrichment, DAS was enriched in structural and functional gene networks of skeletal muscle, resulting in the expression of alternatively spliced transcript isoforms that have been associated with the physiological changes to skeletal muscle in microgravity, including muscle atrophy and altered fiber type function. Finally, RNA-binding proteins, which are required for regulation of pre-mRNA splicing, were themselves differentially spliced but not differentially expressed, an upstream event that is speculated to account for the downstream splicing changes identified in target skeletal muscle genes. Our work serves as the first investigation of coordinate changes in DGE and DAS in large limb muscles across spaceflight. It opens up a new opportunity to understand (i) the molecular mechanisms by which splice variants of skeletal muscle genes regulate the physiological adaptations of skeletal muscle to microgravity and (ii) how small molecule splicing regulator therapies might thwart muscle atrophy and alterations to fiber type function during prolonged spaceflight. The online version contains supplementary material available at 10.1186/s13395-022-00294-9.

随着对载人航天飞行兴趣的增加，理解骨骼肌对微重力的有害生理适应所依据的转录组机制的需求也在增加。虽然微重力诱导的差异基因表达（DGE）已被广泛研究，但差异可变剪接（DAS）对骨骼肌转录组的可塑性和功能状态的贡献尚未在动物模型中进行研究。因此，通过评估航天飞行过程中的DGE和DAS，我们着手对暴露于微重力期间的骨骼肌转录组全貌进行首次综合描述。 利用从暴露于微重力或地面对照条件下9周的30周龄雌性BALB/c小鼠的腓肠肌和股四头肌中分离的总RNA和组织切片，进行了RNA测序、免疫组织化学和形态学分析。 针对微重力，骨骼肌转录组通过DGE和DAS两者进行了重塑。重要的是，虽然DGE显示出可变的基因网络富集，但DAS在骨骼肌的结构和功能基因网络中富集，导致产生与微重力下骨骼肌生理变化相关的可变剪接转录异构体的表达，这些生理变化包括肌肉萎缩和纤维类型功能改变。最后，调节前体mRNA剪接所必需的RNA结合蛋白自身发生了差异剪接但没有差异表达，这一上游事件被推测是导致在目标骨骼肌基因中所发现的下游剪接变化的原因。 我们的工作是对航天飞行过程中大肢体肌肉中DGE和DAS协同变化的首次研究。它为理解以下两点提供了新的契机：（i）骨骼肌基因的剪接变体调节骨骼肌对微重力的生理适应的分子机制；（ii）小分子剪接调节疗法如何在长时间航天飞行期间阻止肌肉萎缩和纤维类型功能的改变。 网络版包含补充材料，可在10.1186/s13395 - 022 - 00294 - 9获取。