UC San Diego

MicroRNAs are a newly-discovered class of short regulatory RNAs that function as sequence-specific guides for the down-regulation of target gene expression. lin-4 was the first microRNA to be discovered, and it has two known targets: lin-14 and lin-28, which are regulated by partial base-pairing of the microRNA to target regions in their 3' untranslated regions (3'UTRs). Chapter 1 of this thesis is devoted to analyzing the regulation of lin-4 biogenesis. MicroRNAs are initially transcribed as long primary transcripts, which are processed by RNAseIII enzymes Drosha and Dicer to liberate the mature microRNA. The expression of mature lin-4 is developmentally controlled such that worms do not express detectable levels of the microRNA, until ̃12 hours of development on food. However, RT-PCR techniques revealed that transcription of lin-4 primary transcripts occurs during the early period when mature microRNA is undetectable, suggesting the existence of a post-transcriptional regulatory mechanism preventing premature processing of these transcripts. A GFP-sensor- based RNAi screen for factors involved in this processing block identified a conserved RRM-domain protein, R05H10.2. Inactivation of R05H10.2 by RNAi causes a post- transcriptional defect in the accumulation of mature lin-4 and induces a dramatic growth defect. However, this growth defect is substantially suppressed in lin-14 and lin-28 mutant worms, revealing a previously undescribed linkage between the heterochronic pathway and organismal growth. Chapter 2 of this dissertation is an analysis of the mechanism of target regulation for lin-4. When my dissertation work commenced, the prevailing model for microRNA function held that target regulation occurred by translational control which left target mRNA levels unaffected. The work detailed in chapter 2 is an analysis of the levels of lin-14 and lin-28 mRNAs undergoing lin-4 mediated regulation in wild-type and lin-4 mutant worms, showing that the target mRNAs are degraded as a result of their interaction with the microRNA. The Pasquinelli lab was among the first groups to report degradation of target mRNAs by microRNA regulation of endogenous targets, and my work with lin-4 was combined with analysis of let-7 target regulation performed by other workers in the Pasquinelli lab, and published in Cell in 2005