Mechanistic investigations of the role of RNA in gene regulation

RNA分子参与基因表达的所有步骤。 该提案描述了有机和生物物理化学的应用，最近的特点涉及短RNA的基因调控机制的研究。 将DNA、RNA或核酸模拟物引入细胞中作为调节生物系统中基因表达的手段已经被化学家深入研究。 1998年，Fire和他的同事报告了一个有趣的观察结果，即将双链RNA（dsRNA）注射到蠕虫中。C. elegans导致同源基因表达的特异性沉默。 这种现象被称为RNA干扰或RNAi，随后被证明涉及短dsRNA分子的RNA降解。 RNAi被认为代表了对病毒dsRNA的细胞反应;然而，以前也知道短细胞RNA参与高等生物体发育的调控。 在发现RNAi之后，从蠕虫、苍蝇和人类中克隆了数百种“微小RNA”（miRNAs）。现在已经清楚的是，大量的miRNAs参与了多种高等生物的基因表达调控。最近，已经表明细菌表达赋予对细菌病毒或噬菌体感染的抗性的短RNA。与如上所述靶向RNA相反，细菌系统似乎靶向外源DNA进行降解。 我们正在研究参与细菌RNA干扰的蛋白质和蛋白质-RNA复合物的结构和功能。 使用合成的RNA底物，我们正在探测干扰途径中发生的蛋白质-RNA相互作用。 高分辨率结构分析将使我们能够确定特定RNA识别的基础和干扰机制。 这些研究将增强我们对催化机制以及蛋白质和RNA结构的理解，并帮助我们进一步阐明小RNA在基因调控中的作用。

RNA molecules are involved in all steps of gene expression. This proposal describes the application of organic and biophysical chemistry to the study of a recently characterized mechanism of gene regulation involving short RNAs. The introduction of DNA, RNA, or nucleic acid mimics into cells has been intensively investigated by chemists as a means to modulate gene expression in biological systems. In 1998, Fire and colleagues reported the fascinating observation that injection of double-stranded RNA (dsRNA) into the worm. C. elegans resulted in a specific silencing of the homologous gene's expression. This phenomenon, termed RNA interference or RNAi, was subsequently shown to involve RNA degradation by short dsRNA molecules. RNAi is believed to represent a cellular response to viral dsRNA; however, it had previously also been known that short cellular RNAs were involved in regulation of development in higher organisms. Subsequent to the discovery of RNAi, several hundred "micro RNAs" (miRNAs) were cloned from worms, flies, and humans. It is now clear that a large number of miRNAs are involved in regulation of gene expression in diverse higher organisms. Recently, it has been shown that bacteria express short RNAs that confer resistance to infection by bacterial viruses or phage. The bacterial system appears to target foreign DNA for degradation as opposed to the targeting of RNA as described above. We are studying the structure and function of protein and protein-RNA complexes involved in bacterial RNA interference. Using synthetic RNA substrates, we are probing the protein-RNA interactions that occur in the interference pathway. High-resolution structural analysis will allow us to determine the bases of specific RNA recognition and the interference mechanism. These studies will enhance our understanding of catalytic mechanisms as well as protein and RNA structure and help us further elucidate the role of small RNAs in gene regulation.