Researchers use parallel analysis of mRNA structure (PARS), ribosome profiling and RNA sequencing (RNA-seq) to reveal that bacterial mRNA structure functions beyond information transfer

Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown.

Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, researchers from the Universities of Potsdam and Hamburg unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage.

PARS analysis.
(A) Overview of modified PARS approach. RNase V1 cleaves double-stranded RNA and combination of RNases A/T1 the single stranded RNA with optimal activities at physiological pH (7.0). RNAse A/T1 usage requires an additional phosphorylation step prior to library generation. (B) The PARS score of the rpoS leader sequence (inset) was overlaid with the experimentally determined structure. Double-stranded nucleotides with positive PARS score are colored red, single-stranded nucleotides with negative PARS score–blue, nucleotides with missing PARS score or equal to zero–green. The color intensity of the rpoS nucleotides reflects the PARS scores (rainbow legend). (C) Metagene analysis of protein-coding transcripts. Average PARS score for each nucleotide (top) and GC content (bottom) across the 5’UTRs, CDS and 3’UTRs of all protein-coding transcripts, aligned at the start or stop codon, respectively. For the shaded areas the average PARS scores or GC content is calculated; thus note the deviations from the total GC content of 51% in E. coli. Unstructured region upstream of the start codon and structured sequence preceding the stop codon are marked by arrows with filled and open arrow heads, respectively.

This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.