microRNAs (miRNAs) play a pivotal role as post-transcriptional regulators in gene expression, adding a layer of complexity to the intricate system of molecular interactions within cells. Despite their short length, miRNAs exhibit remarkable specificity in targeting messenger RNAs (mRNAs), forming specific interaction networks termed the miRNA targetome. Predicting mRNA targets and their repression efficacies remains a significant challenge due to the vast number of potential interaction partners, and the lack of methodologies capable of detecting and extracting biochemical and structural information from these small molecules.

This thesis investigates the structural and functional aspects of miRNA-mediated gene regulation, aiming to elucidate the underlying mechanisms governing miRNA activity via the Argonaute (AGO) protein, which forms the core of the RNA induced silencing complex (RISC). Using the conserved miR-34a and 12 mRNA targets as a model system, we develop and employ various tools including electrophoretic mobility shift assays (EMSA), structural probing techniques, luciferase gene reporter assays, and molecular dynamics simulations, to delineate the biophysical features of miRNA and their interactions.

Our findings demonstrate the importance of the underlying RNA:RNA interactions in determining regulatory outcomes by RISC. We show that the structural binding mode is encoded by the underlying miR-34a:mRNA interaction, independent of the AGO2 protein. Moreover, our analysis suggests that repression efficiency is influenced by the RNA duplex structural class. We observed that miRNA-bulge structures exhibit the strongest repression effect, correlating positively with affinity, whereas mRNA-bulge structures show moderate repression with no significant correlation to affinity. In contrast, symmetrical structures exhibit weak repression effects. Using molecular dynamics simulations, we further illustrate that the novel miRNA-bulge structure can be readily accommodated within the AGO2 protein. We find that AGO2 plays a bidirectional role in modulating the miR 34a:mRNA affinity: weakening strong RNA:RNA binders while enhancing weak binders. Additionally, we observe that miRNA duplex release is more pronounced in high affinity miR-34a:mRNA interactions.

This thesis presents two related projects. Project I describes the methodological developments necessary for achieving the results presented in Project II and has extended applicability to the broader study of RNA structure beyond the scope of this thesis. Project I was published in the RNA Journal (2023). In Project II (manuscript under review), we describe the functional implications of RNA:RNA interactions within the RISC complex, highlighting the impact of the RNA binding mode in determining miRNA-mediated repression and contributing insights into the interplay of biophysical factors determining miRNA function.