Biological functions and post-transcriptional regulation of microRNAs

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

MiRNAs are small noncoding RNAs that are loaded into Argonaute proteins to form the core of the miRNA-Induced Silencing Complex (miRISC). MiRNAs guide miRISC to complementary target mRNAs to silence their expression. Mutations in miRNA loci disrupt gene expression programs, and thus can contribute to the development of various diseases, including cancer. Consequently, understanding both the functions of miRNAs in normal development and the molecular mechanisms that regulate miRNAs are biological questions of critical importance. Understanding the biological functions of miRNAs during embryogenesis While the functions of miRNAs in differentiated tissues are well-studied in C. elegans and other organisms, the embryonic functions of only a few animal miRNAs are understood. Moreover, how miRNAs contribute to the largely post-transcriptional control of gene expression prior to the maternal-to-zygotic transition (MZT) is completely unknown. C. elegans is an excellent model organism in which to study embryonic development due to its well-defined stereotypic cell lineage and powerful genetic tools. Our group is using use C. elegans to elucidate the functions of miRNAs in embryogenesis, first focusing on the miRNA families that are required for embryogenesis. To dissect the biological pathways controlled by these miRNAs, our group is conducting forward (mutagenesis) and reverse (RNAi) screens for suppressors of microRNA family mutant phenotypes. Understanding the biological networks impacted by the embryonically-expressed microRNA families will yield important insights into how gene expression is controlled to coordinate embryogenesis. Defining the molecular mechanisms of miRNA and Argonaute turnover The balance of the rates of miRNA biogenesis and decay control miRNA abundance, and thus gene expression programs. Previous research has carefully elucidated mechanisms of miRNA biogenesis. However, we know very little about how miRNAs and miRISC are turned over either constitutively or in a regulated manner. This is a major gap in our understanding of miRNA regulation, and thus the regulation of gene expression. We are using deep sequencing to assess the contribution of candidate players in microRNA turnover, as well as identify novel effectors and regulators of miRNA turnover. A major unanswered question is whether the miRNA and its protein binding partner Argonaute are targeted for degradation together, or can instead be turned over separately. By measuring the effect of changes in miRNA turnover kinetics on Argonaute turnover kinetics, and vice versa, we are investigating how these events are interconnected. In the long term, we will thus establish a hierarchy of events in the process of miRISC turnover. Understanding the regulation of miRNAs by turnover may lay the foundation for new modalities to treat human disease. This area of study could impact the treatment of cancer, since miRNAs are known to be globally downregulated in many cancer types, and miRNA biogenesis enzymes can act as haploinsufficient tumor suppressors. These data suggest that a modest increase in miRNA abundance could reverse the contribution of miRNAs to cancer. Thus, inhibition of miRNA turnover is an attractive therapeutic strategy. Since enzymes are likely to play a part in this process, small molecules inhibitors of miRNA turnover could be feasible as a targeted cancer therapy. This fiscal period has primarily focused on establishing the infrastructure for carrying out this research program. Start-up funds have allowed us to outfit the lab space with all equipment necessary for C. elegans genetics, cell biology, and molecular biology. We have also begun to establish assays for microRNA turnover and Argonaute turnover rates, and to establish forward screening paradigms for microRNA abundance.