肌肉分化过程中miRNA调控翻译的分子机制

MiRNAs have been shown to play pivotal roles in almost every aspect of biology such as development, differentiation, oncogenesis, and apoptosis. Mature miRNAs exist as ~22nt single stranded RNAs, complexed with one of the Argonaute (Ago) proteins. They are thought to recognize their mRNA targets via limited base pairing between the “seed” region (2-7 nucleotides of the miRNA) and 3’ untranslated regions (3’ UTRs). In contrast, studies conducted in worms without overexpression showed that perfect “seed” base pairing is neither necessary nor sufficient to confer regulation in vivo. Moreover, most of published miRNA-mediated control are quite modest (~2 fold). In our preliminary study, we found conditions in which a subset of miR-1 targets are substantially more regulated than seen with the published (up to 20 fold). To further understand the mechanism of miRNA-mediated gene regulation during muscle differentiation, we propose to 1) identify and characterize of cis-acting elements within these contexts that promote miRNA-mediated regulation; 2) identify potential ancillary factors or identifying modifications, if any, of components of the miRNA machinery to elucidate the underlying mechanism(s) of action; 3) identify endogenous miRNA targets during muscle differentiation.

miRNA参与生命过程的各个方面，包括发育，细胞分化，癌症和细胞调亡等。成熟的miRNA以～22nt的单链RNA存在于Argonaute复合物之中。这些复合物一般被认为通过“种子”序列识别mRNA靶标的3’UTR中的结合位点。但是，“种子”序列并不是决定miRNA靶标的充分必要条件。线虫体内非过表达的实验证实“种子“序列”并不能够决定有效的miRNA调控。除此之外，大多数miRNA介导的调控非常微弱。我们的前期实验找到一些miR-1的靶标能在特定的条件下被miRNA显著调控。为了继续解析肌肉细胞中miRNA介导的基因调控的分子机制，我们计划从一下几个方面开展研究：1）找到和解析促进miRNA功能的环境辅助序列；2）找到促进miRNA功能的蛋白质辅助因子或miRNA调控机器的翻译后修饰，从而进一步解析miRNA调控翻译的分子机制; 3）找到肌肉细胞分化时miRNA的内源靶标。

随着生物复杂度的升高，基因组中非编码序列的比例也相应增大，显示非编码RNA在生命活动与进化过程中具有重要意义。对真核细胞中非编码RNA及其基因的发掘和功能研究，有可能揭示一个全新的由非编码RNA介导的遗传信息传递方式和表达调控网络，从不同于蛋白质编码基因的角度来注释基因组的结构与功能，深入阐明生命活动及遗传与进化的本质。我们在分化肌肉细胞中找到miR-1/206调控的且与多聚核糖体结合的靶标（POGK基因3’UTR）。由于肌肉细胞分化系统更接近miRNA在体内作用时的环境，该靶标与miR-1/206的作用模式为我们提供了一个独特的机会来研究miRNA调控翻译的核心分子机制，miR-1/206是参与的翻译起始后的调控。通过puromycin（翻译延伸链终止剂）确定与多聚核糖体结合的靶标mRNA是否仍然进行有效的翻译延伸（Translation elongation）。我们现在的假设是miRNA所调控的靶标mRNA将要产生的蛋白质是在翻译延伸过程中同时被降解了。在肌肉分化过程中，miRNA的靶标会被特异招募到内质网周围被Argonaute强烈沉默。解析miRNA在体内的重要靶标对理解相关疾病的发病机制有重要意义。为此，我们发展了研究蛋白/核酸互作的新方法GoldCLIP。