Structural and Mechanistic Studies of Regulation of let-7 biogenesis by Lin28'

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DESCRIPTION (provided by applicant): Cancer, stem cell pluripotency, and developmental timing are some of the important biological processes that Lin28 plays a major role in. Lin28 can induce cell reprogramming to pluripotent state when used in combination with OCT4, SOX2, and NANOG. Up-regulation of Lin28 correlates with cell transformation, and many human tumors exhibit high levels of Lin28 (~15%). As part of a feedback loop that involves NF- kB, Lin28 couples inflammation and cell transformation. Moreover, Lin28 variations correlate with variance in human developmental traits such as height and timing of puberty onset. Lin28 is an evolutionarily conserved RNA-binding protein that inhibits the let-7 family of microRNAs. MicroRNAs are small non- coding RNA molecules that regulate specific target-gene expression. In order to generate functional mature microRNAs, the precursor RNA has to undergo processing steps after initial transcription. During this post- transcriptional stage, Lin28 can block both of the cleavage steps (by Drosha and Dicer), and promote degradation by recruiting a terminal uridylyl transferase, specifically for the let-7 microRNAs. Lin28 binds to let-7 precursors, but how these effects are achieved is still unclear. Moreover, although Lin28 activity is specific for let-7 microRNAs, how the various sequences are recognized is also unknown. In order to gain a mechanistic understanding of Lin28 activity, more molecular details are required to explain its specificity and regulation. We will determine high resolution structures of Lin28:let-7 complexes to provide such detailed information. By combining biochemical methods and structural approaches including X-ray crystallography and NMR spectroscopy, we will clarify how Lin28 recognizes its target. Acting upstream of MYC, Lin28 poses as an attractive target for cancer therapeutics. With the help of the structural information, we will perform a high-throughput search for a small molecule inhibitor of Lin28. The effector function of Lin28 will also be examined by investigating how it activates uridylyl transferase on let-7 precursors. Our goal of understanding how Lin28 specifically binds its target, recruits a downstream effector, and can be blocked, are all important steps to better understand let-7 regulation with many biological implications. In addition to its direct impact on controlling let-7 targets, what we learn about Lin28-one of the first post- transcriptional regulators of microRNAs-may be applied to a rapidly growing list of other RNA-binding factors that appear to regulate other miRNAs. Finally, elucidating how Lin28 inhibits microRNA processing will also provide new information on the mechanism of central events in microRNA processing.