The logic and evolution of miRNA strand selection in Caenorhabditis elegans

MicroRNAs (miRNAs) are small RNA molecules that regulate when and where genes are expressed, making them essential for normal development, tissue identity, and the maintenance of healthy cells. Before a miRNA can regulate genes, cells must select one of two possible strands produced from the same precursor molecule. Because each strand recognizes a different set of target genes, this choice fundamentally determines how genes are regulated within individual cells. Despite decades of research on miRNAs, the molecular rules that govern strand selection remain poorly understood. This project will investigate these rules using the microscopic roundworm Caenorhabditis elegans, a powerful experimental model whose gene regulatory pathways are highly conserved with those of humans. By uncovering the fundamental principles that determine miRNA strand selection, this research will advance the understanding of RNA-mediated gene regulation across animals and provide a stronger foundation for future biomedical research and biotechnology development. The project also includes a strong educational component through the RNA Ambassadors program, in which undergraduate students receive training in RNA biology and science communication while developing videos, animations, podcasts, and other educational resources for public audiences. These freely available materials will promote scientific literacy, broaden public appreciation of RNA research, and help prepare the next generation of scientists and science communicators, thereby advancing the national interest through scientific discovery, education, and biotechnology workforce development. This project seeks to establish the first comprehensive mechanistic framework explaining how miRNA strand selection is determined in vivo. The first objective is to define how intrinsic sequence features and thermodynamic properties of miRNA duplexes influence strand choice by systematically engineering miRNA variants and quantitatively measuring strand usage using the HiTmiSS platform, a highly sensitive assay developed by the investigators. The second objective is to identify tissue-specific accessory factors that regulate strand selection by combining Argonaute immunoprecipitation, quantitative proteomics, and targeted genetic perturbations in Caenorhabditis elegans. Together, these complementary experimental approaches will determine how intrinsic RNA properties interact with tissue-specific cellular environments to establish functional miRNA strand preferences. The resulting mechanistic framework will substantially advance understanding of miRNA biogenesis and post-transcriptional gene regulation, improve predictive models of miRNA function, and generate broadly applicable principles that are expected to extend across animal species to advance biotechnology applications. Because miRNAs regulate thousands of genes involved in development, physiology, and disease, the knowledge generated by this project will provide an important conceptual foundation for future investigations of RNA biology and gene regulatory mechanisms. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.