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.
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