Biological functions and post-transcriptional regulation of microRNAs

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

MicroRNAs (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 (reviewed in Kotagama, et al. 2024). 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. We are conducting forward (mutagenesis) and reverse (RNAi) screens for suppressors of microRNA family mutant phenotypes, currently focusing on the deeply conserved mir-51 family. We are also leveraging the power of CRISPR-Cas9-mediated genome editing to discover miRNA-target interactions that are essential to development (Yang, et al. 2020). 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 previously demonstrated that Caffeine-Induced Death (CID-1) is necessary for uridylation of miRNAs, and F31C3.2 (which we named GLD-2 Related-2) is required for adenylation of miRNAs (Vieux, et al. 2021). We also found that these terminal modifications do not play a global role in influencing miRNA decay rates. More recently, we are investigating the regulated decay of the mir-35 family, which is essential for embryogenesis and sharply downregulated thereafter. We recently showed that this miRNA family's decay is dependent upon its seed sequence (nucleotides 2-8), but not other parts of the miRNA sequence (Donnelly, et al. 2022). This represents a novel class of miRNA decay mechanism that may be harnessed therapeutically to modulate abundance of all redundant members of a miRNA seed family simultaneously. Determining the role of Argonaute catalytic activity in animal development The Argonaute proteins that act along with miRNAs to effect gene silencing contain RNAse H-like cleavage activity, but the function of this RNA cleavage activity is very poorly understood in the miRNA pathway in which targets are rarely cleaved. We leveraged C. elegans genetics to determine ancestral roles of the cleavage activity that may exert selective pressure to maintain it over evolutionary time. By creating catalysis-inactivating mutations in the miRNA Argonaute genes by CRISPR, we showed that cleavage activity is required for maturation of the miRISC by aiding in passenger strand destabilization (Kotagama, et al. NAR 2024). Noncanonical biogenesis of microRNAs Most miNAs are generated through two successive endonucleolytic cleavages of a long primary transcript. We recently discovered a new non-canonical pathway that generates miRNAs from fairly short Pol III transcript, and does not require the first endonuclease involved in canonical microRNA biogenesis (Sakhawala, et al. 2025). Understanding non-canonical routes of miRNA biogenesis could enable alternative routes of therapeutic miRNA delivery in diseases in which canonical miRNA biogenesis is incapacitated.