Experimental identification of preQ1-binding RNAs in the pathogenic bacterium Listeria monocytogenes

TL;DR

This study identifies RNA elements in Listeria monocytogenes that bind preQ1, revealing a new riboswitch and a regulatory mRNA element.

Contribution

A novel preQ1 riboswitch and a new mRNA element regulating translation via preQ1-dependent stop codon readthrough are discovered.

Findings

A predicted preQ1 riboswitch from L. monocytogenes binds two preQ1 molecules and regulates translation.

A candidate mRNA (lmo2684) contains a preQ1 riboswitch-like sequence that promotes stop codon readthrough.

Ligand-based pull-down strategies effectively enrich RNAs with undiscovered regulatory functions.

Abstract

Riboswitches are widespread regulatory RNA modules in bacteria, with many different classes already identified and even more yet to be discovered. Traditionally, the identification of riboswitches has relied on bioinformatic analyses and genetic screens. In this work, we explored the possibility of identifying and characterizing predicted and novel riboswitches using an affinity purification-based approach with a functionalized preQ1 ligand. We successfully enriched a predicted preQ1 riboswitch from L. monocytogenes total RNA. Biophysical characterization revealed that this riboswitch can simultaneously bind two ligand molecules and functions as a regulator of translation in vivo. Furthermore, a transcriptome-wide pull-down experiment resulted in strong preQ1-dependent enrichment of several candidate sequences. Characterization of the lmo2684 candidate mRNA revealed a preQ1 riboswitch-like sequence in its 5′ untranslated region. Notably, preQ1 allowed translation of an upstream open reading frame in this region by promoting stop codon readthrough. Our findings highlight the utility of ligand-based pull-down strategies for enriching mRNAs with aptamers that elude computational detection and may possess undiscovered functions.

Pull-down, chemical, and functional analysis of preQ1-binding RNAs: Validation of a predicted preQ1 riboswitch binding two preQ1 molecules and discovery of an mRNA element regulating upstream open reading frame usage in a preQ1-dependent manner.

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