Mechanisms of Action of Gene-Regulatory RNA Molecules

RNA plays a central role in many cellular processes and represents an attractive drug target. Riboswitches are naturally occurring RNA molecules found typically in the 5´-leaders of bacterial messenger transcripts where they support cellular homeostasis by sensing metabolites or ions. Sensing requires a conserved aptamer that binds a cognate ligand, leading to conformational changes in an associated expression platform that control expression of a downstream gene. Although the structures of many riboswitches are known, most are missing their expression platforms, leaving the interplay between aptamer and expression platform unclear. To bridge this knowledge gap, we investigate intact riboswitches using high-resolution structural approaches as well as in- cell methods that reveal functionally relevant RNA conformations in live bacteria. Here, we will broaden the scope of our research using the MIRA mechanism to elucidate the molecular mechanisms of new riboswitches that sense key metabolites and monovalent ions. Many of these ‘class X’ riboswitches are predicted to adopt unique architectures. By contrast, ‘class Y’ riboswitches evolved through mutations in class X sequences, allowing them to retain class X folds but with new ligand specificity. Our preliminary analysis of the class Y spermidine riboswitch revealed a global fold reminiscent of the class X, S-adenosylmethionine (SAM) riboswitch but with substantial structural variations. Biochemical analysis revealed the riboswitch binds two spermidine ligands, suggesting positive cooperativity. We also found that a specific k-turn binding protein greatly enhances spermidine affinity. Given these findings, our future work will focus on the spermidine riboswitch while addressing broader questions in the field: (i) What are the folds of unknown class X & Y riboswitches? How do these folds confer ligand specificity? The spermidine riboswitch reveals mutations that block binding to the SAM ligand while conferring a new ligand preference; (ii) What is the role of cooperativity in riboswitch function? Does biochemical cooperativity confer a more binary (faster) gene-regulatory response? A growing number of riboswitches bind two ligands, prompting us to develop quantitative tools to analyze cooperativity using isothermal titration calorimetry, which will be applied here; (iii) Do k-turn binding proteins enhance ligand affinity for riboswitches and do these proteins affect gene regulatory function? Multiple riboswitches possess conserved k-turns but the role of protein binding in riboswitch function has not been assessed. (iv) What are the molecular determinants of gene regulation? Recently, we discovered a riboswitch class that still achieves significant ligand-dependent gene regulation in cells, without expression platform sequestration. This finding demonstrates the value of our approach, which will relate high-resolution structures to biochemical, computational, and functional data to create a holistic view of class X & Y riboswitch mechanisms of action. Our results are broadly relevant to the creation of RNA biosensors, prediction of RNA structure, and development of RNA-targeted therapeutics. Project Number: 1R35GM161165-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Joseph Wedekind | Institution: UNIVERSITY OF ROCHESTER, ROCHESTER, NY | Award Amount: $427,227 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11257455