In the last decade, microRNAs (miRNAs) have been identified as a prolific class of post-transcriptional regulators of gene expression acting in plants and animals. miRNAs are endogenous, non-coding, 20-24 nt long single-stranded RNAs which suppress target messenger RNAs (mRNAs) either by translational repression or endonucleolytic cleavage. The role of miRNAs in regulating cell division, differentiation, and death makes them essential in development and maintenance of coordinated cell cycle, and their impairment has critical impact on organism health. A number of miRNAs have been implicated in human cancers where their activity as tumor suppressors or oncogenic agents has earned them the term 'oncomirs.'Members of the let-7 class of oncomirs in particular act as tumor suppressors, and decreased levels of its transcripts are correlated with a poor prognosis in lung and breast cancers. While much is known mechanistically about their biogenesis and maturation, the regulation and degradation of miRNAs has not been described. The proposed work is a structure-function analysis of a precursor-miRNA, pre-let-7g, in complex with a regulatory RNA-binding protein, Lin28. Lin28 has been found highly expressed in certain cancer cell lines and its downregulation of let-7 plays an important role in tumorigenesis.
Specific aims of the proposed work include the (i) low and high resolution structural studies of pre-let-7g, Lin28, and the bound complex by solution NMR (ii) identification of the interface of binding and biochemical and/or kinetic characterization of the pre-let-7g/Lin28 association, and (iii) validation of Lin28- recognized structural elements in a human breast cancer cell line. In combination, solution NMR methods and biochemical techniques will provide structure and dynamics data which will yield a more integrated understanding of the regulatory pair in vivo, and provide the basis for a model of recognition of let-7 by Lin28. We hypothesize that a targeted inhibition of the let-7/Lin28 binding, informed by our structural and biochemical characterizations, could reverse the trend of proliferation in a chosen breast cancer cell line. This validation could represent the pilot study for more directed searches in future cancer therapeutics design. The solution structure of the pre-let-7g/Lin28 complex, which would represent the first such structure for a miRNA in complex with a regulatory protein, will also significantly advance our understanding of general miRNA regulation mechanisms.
The proposed work aims to structurally characterize a regulatory miRNA-protein interaction that has been implicated in human lung, breast, urothelial, and cervical cancers. Rational drug design, which relies heavily on structural data input, could be directed towards an inhibition of this interaction in order to rescue the native tumor-suppressor effects of the miRNA of interest, let-7g. Targeting the maturation and regulation pathways of miRNAs, a broad class of molecules, could result in a new category of cancer therapeutics.