Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Gene expression, the genetic information flow from DNA to RNA to protein, involves delicate regulation by a group of small non-coding RNAs called microRNAs (miRNAs). Dysregulated miRNA networks have been observed in a variety of disease states including cancer, developmental defects, and neurodegenerative diseases. In these disease states, the miRNA abundance can be affected at either the degradation or the biogenesis steps. Target-directed miRNA degradation (TDMD) is a mechanism by which specific miRNAs undergo degradation when extensively base-paired with their target RNAs. How TDMD may contribute to different diseases is unclear due to the paucity of known target RNAs (or triggers) that can induce miRNA degradation. On the other hand, Dicer functions as the central enzyme in processing precursor miRNAs into mature miRNAs. Dicer deficiency promotes tumorigenesis, but how different miRNAs are affected by lower levels of Dicer in cancer remains a question. To address these knowledge gaps, we will probe the molecular regulatory mechanisms of specific miRNA abundance in metazoans focusing on three projects: 1. Expand the repertoire of metazoan TDMD triggers. We have established CLASH, a combined biochemical and bioinformatics pipeline, for identification of TDMD triggers in cultured cells. We will further improve CLASH for application in model organisms to identify conserved and essential TDMD triggers. 2. Investigate the properties of effective TDMD triggers. Known TDMD trigger sequences are concentrated in conserved regions of the mRNA 3′ UTR and non-coding RNAs. We will investigate the importance of trigger transcripts sequences, structure and the non-coding properties in inducing TDMD. 3. Differential Dicer processing of cellular miRNAs. In an engineered colorectal cancer HCT116 cell line, in which Dicer expression can be fine-tuned by doxycycline, we will determine the miRNA profile at different Dicer expression levels. Determinants for differential responsiveness to Dicer of individual miRNAs will be probed by in vitro Dicer cleavage assays and identification of miRNA binding proteins. Our unique experimental approaches will provide the basis for developing novel therapeutics through modulation of the TDMD pathway and Dicer action. Techniques and experience acquired in this project will prepare our group for future research in exploring molecular machineries that regulate small RNAs essential for gene expression in normal and diseased tissue.
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