Comparison of 5' UTR structures in Listeria monocytogenes

To understand physiological processes in vivo, it is vital to disclose the conformation of biological molecules inside living cells and correlate their structure with their function. Here, we describe a method, FUSE (5'-UTR Structure Elucidation) that was used to characterize changes in the structures of 5'-UTRs at different conditions inside the human bacterial pathogen Listeria monocytogenes. Using FUSE, we discovered a novel RNA thermoswitch and calculated the ratio of two conformations that this thermoswitch assumes at different conditions. FUSE could also identify the site of base-pairing interaction between a small RNA and mRNA with a single-nucleotide resolution. Strikingly, FUSE discovered a functional mRNA-mRNA interaction, where the stability of mRNA encoding PrsA2 chaperone, was increased by the direct binding of an mRNA encoding its substrate, listeriolysin O, the main L. monocytogenes virulence factor. Owing to its focus on 5'-UTRs, FUSE can be applied to analyse these regulatory regions in any living organism. Overall design: L. monocytogenes EGD-e cells or RNA isolated from these cells were treated with dimethyl sulphate (DMS). The modified RNA served as a template for cDNA library construction according to FUSE (5' UTR Structure Elucidation) or Structure-seq protocols. Both these methods employ TGIRTIII reverse transcriptase to encode methylated adenines and cytosines as mismatches in cDNA sequence. While Structure-seq protocol synthesizes cDNA from the total RNA, FUSE selectively enriches for the 5' UTR regions in the cDNA library. The resulting cDNA libraries were sequenced on Illumina MiSeq platform in 2 x 75 bp paired-end mode.