Biogenesis and function of the small temporal RNA let-7

描述（由申请人提供）：两类相关的21- 23nt RNA——小干扰RNA （sirna）和小时间RNA （stRNAs）——为mRNA表达的转录后控制提供了核酸特异性决定因素，sirna是双链的，在RNA干扰（RNAi）途径中作用于靶mRNA进行核内裂解，通过触发mRNA破坏来沉默其表达。相反，stRNAs是单链的，被认为在不改变mRNA稳定性的情况下调节mRNA翻译。目前的证据表明，尽管RNAi和stRNA的靶基因调控模式不同，但它们的途径却非常相似。事实上，多结构域RNase III酶Dicer是生成sirna和strna所必需的。Dicer切割长双链RNA以产生介导RNAi的sirna，而它作用于小的（约70 nt）茎环前体RNA以产生stRNAs。Dicer如何产生stRNAs以及为什么这些stRNAs介导翻译控制而不是通过RNAi途径介导mRNA降解仍未被发现。此外，缺乏一个体外系统来概括stRNAs的翻译控制，这阻碍了理解它们调节基因表达的生化机制的努力。本文提出的实验旨在定义stRNA产生的生化机制，确定siRNA和stRNA命运的区别，并验证旨在解释stRNA与3' UTR序列结合如何抑制mRNA翻译的特定假设。具体来说，实验被提出：(1)鉴定从其72nt前体茎环RNA中产生stRNA let-7所需的蛋白质；(2)确定成熟的let-7不对称产生所需的pre- 7 RNA的序列或结构特征；(3)确定为什么stRNAs不触发其靶mrna的切割；(4)重组pre- strna以生成功能性sirna而非strna；(5)研究stRNAs调控其靶mRNA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.