# Mechanisms of Action of Non-Coding RNA Molecules

> **NIH NIH R01** · UNIVERSITY OF ROCHESTER · 2020 · $377,671

## Abstract

Abstract. RNA plays a key role in most aspects of gene regulation, and is increasingly viewed as a drug
target. To fully exploit RNA in medicine and biology, innovative approaches are needed to probe the
interactions between structured RNAs and small molecules with the goal of elucidating how binding alters
cellular function. Riboswitches are natural RNA sensors located typically in the 5´-leader of mRNAs where they
bind cognate effectors to regulate downstream genes. We previously determined representative crystal
structures of the three known preQ1 riboswitch classes revealing: new tertiary folds, novel determinants of
ligand recognition, and diverse strategies to bury associated Shine-Dalgarno Sequences (SDSs). Nonetheless,
a substantial gap still exists in the field's understanding of the underlying signal transduction pathways that
connect effector binding to gene-regulatory conformation. To address this formidable challenge, we developed
new tools to cogently relate structure to function: (i) we prepared a robust bacterial reporter in which a preQ1
riboswitch represses GFPuv expression in response to the effector preQ1 (EC50 6.9 nM); (ii) we developed
SiM-KARTS (Single Molecule Kinetic Analysis of RNA Transient Structure), which detects effector-induced
conformational changes by monitoring repeated association and dissociation events between the riboswitch
SDS and a probe mimicking the 3´-end of 16S ribosomal RNA. Our results show that SDS exposure occurs in
`bursts' that diminish with preQ1; (iii) we developed 2-methylnicotinc acid imidazolide (NAI) and 3-nicotinoyl
azide (NAz) to probe specific preQ1 riboswitch conformations that regulate the in cell GFPuv reporter, whose
control status is visualized by fluorescence. These novel approaches form a strong basis to investigate the
premise that discrete signal transduction networks link effector binding to riboswitch gene regulation. We will
address this overarching goal in three dovetailed aims: (Aim 1) Identify preQ1 riboswitch mutants integral to
signal transduction; (Aim 2) Quantify SDS accessibility and dynamics of riboswitch variants; and (Aim 3) Relate
riboswitch biophysical data to in vivo chemical modification analysis to elucidate interaction networks operative
in gene regulation. To our knowledge, no other group is using such tools and approaches to dissect effector-
mediated signal-transduction pathways for an entire riboswitch family. We are a team of experts comprising
four P.I.s with strong records in: RNA structural biology, effector binding, RNA mutagenesis, and biophysical
studies (Wedekind, P.I.); single-molecule FRET and methods development (Walter, U. Michigan); RNA probing
reagents, protocols, and analysis (Spitale, UC Irvine); and `RNAstructure' prediction using experimental
restraints (Mathews, University of Rochester). We are uniquely qualified to perform this research. High-value
outcomes include the unprecedented elucidation and in vivo validation of signal transduc...

## Key facts

- **NIH application ID:** 10000980
- **Project number:** 5R01GM063162-16
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** Joseph E Wedekind
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $377,671
- **Award type:** 5
- **Project period:** 2001-04-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10000980

## Citation

> US National Institutes of Health, RePORTER application 10000980, Mechanisms of Action of Non-Coding RNA Molecules (5R01GM063162-16). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10000980. Licensed CC0.

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