Biogenesis and function of the small temporal RNA let-7

描述（由申请人提供）：两类相关的21-23 nt RNA--小干扰RNA（siRNA）和小时间RNA（stRNA）--为mRNA表达的转录后控制提供核酸特异性决定簇，siRNA是双链的，并在RNA干扰（RNAi）途径中作用以靶向mRNA进行核酸内切裂解，通过触发mRNA破坏来沉默其表达。相比之下，stRNA是单链的，并且被认为调节mRNA翻译而不改变mRNA稳定性。目前的证据表明，尽管它们的靶基因调控模式不同，但RNAi和stRNA途径非常相似。事实上，多结构域RNase III酶Dicer是产生siRNA和stRNA所必需的。Dicer切割长的双链RNA以产生介导RNAi的siRNA，而它作用于小的（约1000个）siRNA。70 nt）茎环前体RNA以产生stRNA。Dicer如何产生stRNA以及为什么这些stRNA通过RNAi途径介导翻译控制而不是mRNA降解仍然未被发现。此外，缺乏一个体外系统，重演stRNA的翻译控制，阻碍了努力理解的生化机制，他们调节基因表达。本文提出的实验试图定义stRNA产生的生化机制，以确定siRNA和stRNA命运的区别，并测试试图解释与3' UTR序列结合的stRNA如何抑制mRNA翻译的特定假设。具体地，提出实验以（1）鉴定从其72 nt前体茎环RNA产生stRNA let-7所需的蛋白质;（2）确定成熟let-7的不对称产生所需的pre-let-7 RNA的序列或结构特征;（3）确定为什么stRNA不触发其靶mRNA的切割;（4）重新设计pre-stRNA以产生功能性siRNA而不是stRNA;以及（5）研究stRNA调节其mRNA靶标表达的机制。

DESCRIPTION (provided by applicant): Two related classes of 21-23 nt RNAs -- small interfering RNAs (siRNAs) and small temporal RNAs (stRNAs) -- provide nucleic acid specificity determinants for the post-transcriptional control of mRNA expression, siRNAs are double-stranded and act in the RNA interference (RNAi) pathway to target mRNAs for endonucleolytic cleavage, silencing their expression by triggering mRNA destruction. In contrast, stRNAs are single-stranded and are thought to regulate mRNA translation without altering mRNA stability. Current evidence suggests that despite their different modes of target gene regulation, the RNAi and stRNA pathways are remarkably similar. In fact, the multi-domain RNase III enzyme, Dicer, is required to generate both siRNAs and stRNAs. Dicer cleaves long, double-stranded RNA to generate siRNAs that mediate RNAi, whereas it acts on small (ca. 70 nt) stem-loop precursor RNAs to produce stRNAs. How Dicer generates stRNAs and why these stRNAs mediate translational control rather than mRNA degradation via the RNAi pathway remains undiscovered. Furthermore, the absence of an in vitro system that recapitulates translational control by stRNAs has hamstrung efforts to understand the biochemical mechanism by which they regulate gene expression. The experiments proposed here seek to define the biochemical mechanism by which stRNAs are generated, to determine what differentiates the siRNA and stRNA fates, and to test specific hypotheses that seek to explain how an stRNA bound to a 3' UTR sequence represses mRNA translation. Specifically, experiments are proposed to (1) identify the proteins required for the production of the stRNA let-7 from its 72 nt precursor stem-loop RNA; (2) to determine the sequence or structural features of pre-let-7 RNA required for the asymmetric production of mature let-7; (3) to determine why stRNAs do not trigger cleavage of their target mRNAs; (4) to re-engineer pre-stRNAs to generate functional siRNAs instead of stRNAs; and (5) to investigate the mechanism by which stRNAs regulate expression of their mRNA targets.