Maturation of let-7 miRNAs

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MicroRNAs (miRNAs) play important roles in the regulation of gene expression by recognizing messenger RNAs (mRNA) through sequence complementarity as part of the RNA-induced silencing complex (RISC). Typically, targeting an mRNA by formation of the miRNA-guided RISC leads to translational arrest and degradation of the mRNA. In humans, the genome encodes around 2,000 miRNAs, which have the potential to regulate over 60% of all mRNAs. Misregulation of miRNA levels can change gene expression patterns, and these changes have been directly linked to developmental defects and several human diseases. The miRNAs of the let-7 family are highly conserved across animal species and play several important roles in many biological processes. Given the key role of let-7 miRNAs as development regulators and tumor suppressors, their biogenesis is tightly regulated. This starts with transcription by RNA polymerase II of a primary miRNA (pri-miRNA), which undergoes two cleavage steps, first by the Drosha/DGCR8 (Microprocessor) complex to yield the precursor miRNA (pre-miRNA) and then by Dicer to yield a miRNA duplex. A number of recent studies have led to the general view that numerous post-transcriptional mechanisms regulate miRNA maturation and that this allows for specific temporal and spatial expression of miRNAs. Moreover, several proteins have been identified that target immature forms of let-7 miRNAs (pri-let-7 and pre-let-7) to control their maturation, including TRBP, Lin28 (Lin28A and Lin28B), Musashi1, TUT4, hnRNPA1 and KHSRP proteins. However, much remains to be known about the mechanistic details of these interactions and how they regulate miRNA maturation. The long-term objective of our research program is to gain a detailed understanding of specific mechanisms that regulate let-7 miRNA maturation. Our working hypothesis is that miRNA maturation requires a complex interplay of protein/protein and protein/RNA interactions that target the pri- and pre-miRNAs to either stimulate or repress the activity of the two main miRNA processing enzymes, Drosha and Dicer. Our current focus is on a subset of proteins known to play critical functions in the regulation of let-7 miRNA levels. In the next five years, we will characterize the mechanistic details by which these proteins regulate the maturation of the let-7 miRNAs at both the molecular and atomic levels. Our work will involve biochemical, biophysical and structural characterization of select protein/protein and protein/RNA complexes that regulate the Dicer and/or Drosha cleavage steps of miRNA maturation. Our studies will provide critical and timely insights into our understanding of miRNA maturation and how miRNA levels are controlled. In addition, given that miRNAs have enormous potential for medical and agricultural applications, our basic research will provide crucial information needed to help evaluate and exploit these new cutting-edge applications.