Effects of siRNA depletion on gene expression during C2C12 skeletal muscle differentiation initiation

In humans, skeletal muscles comprise nearly 40% of total body mass, which is maintained throughout adulthood by a balance of muscle protein synthesis and breakdown. Cellular amino acid (AA) levels are critical for these processes, and mammalian cells contain transporter proteins that import AAs to maintain homeostasis. Until recently, the control of transporter regulation has largely been studied at the transcriptional and post-translational levels; however, our recent work demonstrates that the RNA-binding protein YBX3 is critical to intracellular sustain AA in human cells. Here we report that YBX3 post-transcriptionally controls the levels of specific AA transporter messenger (m)RNAs in skeletal muscle cells, which impacts the intracellular levels transported by these proteins. Further, we find that reduction of YBX3 protein reduces proliferation during skeletal muscle differentiation, and that YBX3 expression increases substantially during skeletal muscle differentiation, which is independent to changes in itsmRNA levels. Taken together, our findings suggest that YBX3 regulates AA transport in skeletal muscle cells and that its expression is critical to maintain skeletal muscle cell proliferation during differentiation. Overall design: To identify genes whose expression is controlled by YBX3, we used high-throughput RNA sequencing (RNAseq) in C2C12 mouse skeletal muscle cells depleted of YBX3 by small interfering RNA (siRNA). We assessed changes in transcripts levels in myoblasts before (proliferating) and one day after differentiation initiation (differentiating), which allowed for cells to be harvested concurrently at 48 hours post siRNA transfection.

在人体中，骨骼肌约占总体重的40%，成年后其质量稳态通过肌肉蛋白质合成与分解的动态平衡得以维持。细胞内氨基酸（amino acid，AA）水平对这一过程至关重要，而哺乳动物细胞携带负责转运氨基酸的载体蛋白，以维持细胞内稳态。直至近期，针对该类转运蛋白调控机制的研究大多集中于转录和翻译后水平；然而我们的最新研究表明，RNA结合蛋白YBX3对人类细胞内氨基酸的稳态维持具有关键作用。

本研究发现，YBX3可通过转录后调控骨骼肌细胞中特定氨基酸转运蛋白的信使RNA（messenger RNA，mRNA）水平，进而影响这些载体蛋白介导的细胞内氨基酸转运过程。进一步研究显示，敲低YBX3蛋白会抑制骨骼肌分化进程中的细胞增殖；而在骨骼肌分化过程中，YBX3的表达量会显著上调，且该变化与其mRNA水平的改变无关。

综上，我们的研究结果表明，YBX3可调控骨骼肌细胞内的氨基酸转运过程，其表达对维持分化阶段骨骼肌细胞的增殖能力至关重要。

整体实验设计：为鉴定受YBX3调控的基因，我们利用小干扰RNA（small interfering RNA，siRNA）敲低C2C12小鼠骨骼肌细胞中的YBX3表达，并通过高通量RNA测序（RNA sequencing，RNA-seq）分析基因表达变化。我们分别在成肌细胞增殖期以及分化启动1天后（此时可与siRNA转染后48小时同步收获细胞）检测转录本水平的变化。