MicroRNAs are small noncoding RNAs that post-transcriptionally reduce protein output from most human mRNAs by mechanisms that are still obscure. This thesis provides insights into three aspects of microRNA biogenesis and function described below. MicroRNA precursors are excised from primary transcripts by the Microprocessor complex containing Drosha and DGCR8. Although most microRNAs are located in introns of protein-coding and noncoding genes, the mechanisms coordinating microprocessing and splicing are unclear. MiR-211 is a microRNA expressed from intron 6 of melastatin, a suspected melanoma tumor suppressor. We demonstrate that miR-211, and not melastatin, is responsible for the tumor suppressive function of this locus, that Drosha-mediated processing of the miR-211 precursor promotes splicing of melastatin exon 6-exon 7 junctions, and that perturbing 5' splice site recognition by the U1 snRNP reduces Drosha recruitment to intron 6 specifically and intronic microRNA levels globally. Thus we identify a novel physical and functional coupling between microprocessing and splicing. Typically, Agos stabilize mature microRNAs and as a complex stoichiometrically bind to complementary mRNAs. We demonstrate an alternative order of events in which Agos bind and repress pre-formed imperfect microRNA-mRNA duplexes in processing bodies of live cells, and cleave pre-formed perfect microRNA-mRNA duplexes in vitro. Our data support a novel catalytic model whereby Agos first deposit microRNAs onto mRNAs and dissociate, thus priming multiple microRNA-mRNA duplexes for concurrent repression by a single Ago. Despite key roles in development and pathogenesis, effectors and regulators of microRNA-mediated repression are still poorly characterized. An RNAi screen revealed that depletion of ribosomal proteins of either small or large ribosomal subunit dissociates microRNA-containing complexes from mRNAs repressed at translation initiation, increasing their polysome association, translation, and stability relative to untargeted mRNAs. Thus ribosomal proteins globally regulate microRNA function. Another RNAi screen revealed that Akt3 phosphorylates Ago2, which negatively regulates cleavage and positively regulates translational repression of microRNA-targeted mRNAs. Thus Ago2 phosphorylation is a molecular switch between its mRNA cleavage and translational repression activities. The following pages will place these novel insights into biological and disease-relevant context, will describe what was known prior to these studies, and will provide perspectives for future studies.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10307759 |
| Date | January 2012 |
| Creators | Janas, Maja |
| Contributors | Novina, Carl D. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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