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

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.