Satellite cell expansion is mediated by P-eIF2α-dependent Tacc3 translation

Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo. Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α-dependent read-through of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α-dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5′ UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α-dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle. Satellite cells of adult Pax3GFP/+ mice after 4-day culture in presence of 10 μM sal003 (4 replicates) or under normal conditions (DMSO, 4 replicates) for a total of 8 samples.

基因表达的翻译调控是成体干细胞静息、激活与自我更新的重要调控机制。在骨骼肌中，静息卫星细胞维持低水平的蛋白质合成，这一过程部分由真核翻译起始因子2α（eukaryotic translation initiation factor 2α, eIF2α）的磷酸化形式（P-eIF2α）介导。通过小分子化合物sal003对eIF2α磷酸酶进行药理学抑制，可维持P-eIF2α的水平，并支持卫星细胞的体外扩增。矛盾的是，P-eIF2α还能增强特定mRNA的翻译，这一过程依赖于P-eIF2α介导的抑制性上游开放阅读框（upstream open reading frames, uORFs）通读。本研究旨在探究P-eIF2α依赖的mRNA翻译是否可支持卫星细胞的扩增。通过转录组学与蛋白质组学分析，我们发现，在经sal003处理扩增的卫星细胞中，多个与纺锤体极组装相关的基因在蛋白质水平上调，而mRNA水平无相应变化。我们证实，有丝分裂纺锤体稳定性基因Tacc3的mRNA 5'非翻译区（5' untranslated region, 5' UTR）中的uORFs可介导P-eIF2α依赖的翻译。TACC3缺失的卫星细胞会出现扩增、自我更新及骨骼肌再生功能缺陷。本研究的样本来源于成年Pax3GFP/+小鼠的卫星细胞，将其分别在添加10 μM sal003的培养基中培养4天（4个生物学重复），以及在正常培养条件（添加二甲基亚砜dimethyl sulfoxide, DMSO）下培养4天（4个生物学重复），共计8个样本。