DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli

Abstract

SummaryThe 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome.

Silencing cryptic specialized metabolism inStreptomycesby the nucleoid-associated protein Lsr2

“…In the proteobacteria, hns expression is activated by multiple transcription factors (Falconi et al, 405 1996;La Teana et al, 2006), and is negatively regulated by both small RNAs and H-NS itself (Falconi et 406 al., 1993;Lalaouna et al, 2015). In the actinobacteria, there is currently nothing known about the 407 transcriptional regulation of lsr2, although it likely governs its own expression: there is a large Lsr2 408 binding site that overlaps the lsr2 promoter (Table S3), suggesting that, like H-NS, it negatively regulates 409 its own expression.…”

The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs

“…Thus, part of these 5′UTRs is overlooked. In the same vein, the coding sequence beyond the first 100 nt after the first nucleotide of the start codon and the 3′UTR are not taken into account, and some sRNAs are indeed pairing deep in the coding sequence (Gutierrez et al, ; Lalaouna, Morissette, Carrier, & Massé, ; Papenfort, Sun, Miyakoshi, Vanderpool, & Vogel, ). Finally, CopraRNA concentrates on protein‐coding mRNAs, that is, noncoding transcripts such as other sRNAs or tRNAs are not yet considered.…”

A game of tag: MAPS catches up on RNA interactomes

“…23 A single sRNA can even adopt different mechanisms of action depending on the mRNA targeted. 25,26 All these characteristics prevent straightforward in silico target prediction. Usually, available bioinformatic programs yield a large pool of false positives and true targets may not present the most intuitive base-pairing pattern.…”

“…DsrA MAPS data prompted us to reinvestigate this RNA-RNA interaction further, leading to the confirmation that rbsD was indeed negatively regulated by DsrA, as the sRNA induces mRNA decay following basepairing. 26 Recently, we also applied MAPS to tRNA-derived RNA fragments in order to determine their binding partners. Deriving from pre-tRNA transcript, tRF proved to be a challenge in accomplishing MAPS experiment due to their specific maturation processes.…”

“…Results obtained for the sRNA DsrA were particularly exciting. 26 DsrA is a well-known effector of positive and negative expression of rpoS and hns mRNA, respectively. [33][34][35][36] As expected, we first validated that both mRNA targets were co-purified with MS2-DsrA.…”