microRNAs are critical regulators of cellular proliferation, differentiation and response to stress but their biogenesis is poorly understood. For example, transcription rates of microRNAs often do not correlate with the level of the corresponding mature microRNA levels, suggesting that post- transcriptional events determine the fate of microRNAs during these important cellular processes. Given that microRNAs are deregulated in human diseases, obtaining a comprehensive mechanistic view of microRNA biogenesis could greatly expand the potential therapeutic opportunities of microRNAs. We recently discovered two new mechanisms regulating the microRNA pathway at the post- transcriptional level. The first mechanism is through post-transcriptional modification of microRNAs. We determined that the BCDIN3D RNA methyltransferase methylates specific microRNA precursors to inhibit their processing into mature microRNAs by Dicer. We further identified RNA and protein partners of BCDIN3D, which revealed new RNA modifications of specific precursors of let-7, a crucial microRNA family involved in cell differentiation and transformation. Our results suggest that a combination of RNA modifications may act in concert to suppress mature let-7 biogenesis. The second mechanism is through a crosstalk between the piwi and micro RNA pathways. Using quantitative proteomics of the proteins interacting with Enoxacin, a small molecule shown to stimulate the microRNA pathway and inhibit growth of transformed cells, we identified the PIWIL3 protein as a primary target of this drug. PIWIL3 is an Argonaut protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNA interference through piwiRNAs. Our results reveal an unexpected link between PIWIL3 and the microRNA pathway that could be crucial for promoting transformation. The above-described processes are novel and previously uncharacterized. This proposal builds upon our findings to decipher the molecular mechanisms that mediate down-regulation of the microRNA pathway by RNA modifications and piwi/microRNA biogenesis crosstalk. To elucidate these mechanisms, we will employ a multifaceted approach using our expertise in cellular and molecular biology, RNA biochemistry, mass spectrometry and next generation sequencing. We expect the findings acquired from this proposal to provide critical new insights into the causes and consequences of microRNA pathway deregulation in human cells that can be leveraged to improve the diagnosis and treatment of human diseases. 1
The proposed research aims to unravel the molecular mechanisms that lead to microRNA pathway deregulation, which is a hallmark of many human pathologies, including heart disease, neurodegeneration, diabetes, viral infection, and cancer. Our identification of RNA modifications that post-transcriptionally regulate microRNA biogenesis, as well as the discovery of a crosstalk between the micro- and piwi-RNA pathways in transformed cells, provide key new insights into both normal and altered microRNA function and regulation. This project is well aligned with the mission of NIH because the knowledge obtained from its successful completion can be leveraged to improve the diagnosis and treatment of human diseases. 1
