David Lalaouna

Regulatory RNAs and target mRNA decay in prokaryotes.

Recent advances in prokaryote genetics have highlighted the important and complex roles of small regulatory RNAs (sRNAs). Although blocking mRNA translation is often the main function of sRNAs, these molecules can also induce the degradation of target mRNAs using a mechanism that drastically differs from eukaryotic RNA interference (RNAi). Whereas RNAi relies on RNase III-like machinery that is specific to double-strand RNAs, sRNA-mediated mRNA degradation in Escherichia coli and Samonella typhimurium depends on RNase E, a single-strand specific endoribonuclease. Surprisingly, the latest descriptions of sRNA-mediated mRNA degradation in various bacteria suggest a variety of previously unsuspected mechanisms. In this review, we focus on recently characterized mechanisms in which sRNAs can bind to target mRNAs to induce decay. These new mechanisms illustrate how sRNAs and mRNA structures, including riboswitches, act cooperatively with protein partners to initiate the decay of mRNAs. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Regulatory RNAs Involved in Bacterial Antibiotic Resistance

An increasing number of RNAs have been recently shown topossess regulatory functions similar to those of proteins. Inbacteria, these regulatory RNAs are usually noncoding and areshort size (50–500 nts) transcripts that are often referred to assmall RNAs (sRNAs) [1,2]. These sRNAs are synthesized underspecific environmental conditions and play a major role in theregulation of various cellular processes (Figure 1) [3]. Most ofthem act via an imperfect antisense base-pairing with their targetmRNAs. Duplex formation usually results in inhibition orstimulation of mRNA target translation. In some cases, sRNAscan also bind proteins to influence their activities (e.g., 6S RNA).Compared to protein-dependent mechanisms, sRNAs require lessenergy, act faster and also allow a coordinated regulation ofseveral targets. Owing to these characteristics, sRNAs allowefficient adaptation of bacteria to their ever-changing environ-ment. Therefore, the possibility exists that some sRNAs may beinvolved in antibiotic resistance. In this report, we provideevidence that illustrates the growing number of sRNAs thatinfluence bacterial resistance to antibiotics.

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

The 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.

Identification of sRNA interacting with a transcript of interest using MS2-affinity purification coupled with RNA sequencing (MAPS) technology

RNA sequencing (RNAseq) technology recently allowed the identification of thousands of small RNAs (sRNAs) within the prokaryotic kingdom. However, drawing the comprehensive interaction map of a sRNA remains a challenging task. To address this problem, we recently developed a method called MAPS (MS2 affinity purification coupled with RNA sequencing) to characterize the full targetome of specific sRNAs. This method enabled the identification of target RNAs interacting with sRNAs, regardless of the type of regulation (positive or negative), type of targets (mRNA, tRNA, sRNA) or their abundance. We also demonstrated that we can use this technology to perform a reverse MAPS experiment, where an RNA fragment of interest is used as bait to identify interacting sRNAs. Here, we demonstrated that RybB and MicF sRNAs co-purified with internal transcribed spacers (ITS) of metZ–metW–metV tRNA transcript, confirming results obtained with MS2-RybB MAPS. Both raw and analyzed RNAseq data are available in GEO database (GSE66517).

The spectrum of activity of the small RNA DsrA: not so narrow after all

For a long time, the small regulatory RNA DsrA has been considered as a regulator with a narrow spectrum of action due to its restricted targetome. Since the first reports on DsrA characterization, only two targets of DsrA have been described: rpoS and hns mRNAs, encoding the sigma factor σS and the nucleoid-associated protein H-NS, respectively. Recently, the scope of DsrA targetome has been expanded by the characterization of two negatively regulated mRNAs, mreB and rbsD, involved in cell wall biosynthesis and ribose metabolism, respectively. In this review, we summarize new insights in DsrA-mediated regulation and emphasize the versatility of DsrA modes of action.

Identification of unknown RNA partners using MAPS

Recent advances in high-throughput sequencing have led to an explosion in the rate of small regulatory RNAs (sRNAs) discovery among bacteria. However, only a handful of them are functionally characterized. Most of the time, little to no targets are known. In Lalaouna et al. (2015), we proposed a new technology to uncover sRNAs targetome, which is based on the MS2-affinity purification (MAPS). We were able to prove its efficiency by applying it on well-characterized sRNAs of Escherichia coli. Thereafter, we adapted the procedure to other kind of RNA (mRNAs and tRNA-derived RNA fragments) and bacteria (pathogenic or Gram-positive strains). Here, we clearly report all improvements and adjustments made to MAPS technology since it was originally reported.

Every little piece counts: the many faces of tRNA transcripts

For over half a century, tRNAs have been exclusively known as decoders of genomic information. However, recent reports evidenced that tRNA transcripts are also bearers of functional RNAs, which are able to execute various tasks through an array of mechanisms. Here, we succinctly review the diversity and functions of RNAs deriving from tRNA loci.

A game of tag: MAPS catches up on RNA interactomes

ABSTRACT In the last few decades, small regulatory RNA (sRNA) molecules emerged as key regulators in every kingdom of life. Resolving the full targetome of sRNAs has however remained a challenge. To address this, we used an in vivo tagging MS2-affinity purification protocol coupled with RNA sequencing technology, namely MAPS, to assemble full bacterial small RNAs targetomes. The impressive potential of MAPS has been supported by a number of reports. Here, we concisely overview RNA-tagging history that preceded the development of the MAPS assay and expose the range of possible uses of this technology.

Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli

Abstract Small RNAs are key components of complex regulatory networks. These molecules can integrate multiple cellular signals to control specific target mRNAs. The recent development of high-throughput methods tremendously helped to characterize the full targetome of sRNAs. Using MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we reveal the targetomes of two sRNAs, CyaR and RprA. Interestingly, both CyaR and RprA interact with the 5′-UTR of hdeD mRNA, which encodes an acid-resistance membrane protein. We demonstrate that CyaR classically binds to the RBS of hdeD, interfering with translational initiation. We identified an A/U-rich motif on hdeD, which is bound by the RNA chaperone Hfq. Our results indicate that binding of this motif by Hfq is required for CyaR-induced degradation of hdeD mRNA. Additional data suggest that two molecules of RprA must bind the 5′-UTR of hdeD to block translation initiation. Surprisingly, while both CyaR and RprA sRNAs bind to the same motif on hdeD mRNA, RprA solely acts at the translational level, leaving the target RNA intact. By interchanging the seed region of CyaR and RprA sRNAs, we also swap their regulatory behavior. These results suggest that slight changes in the seed region could modulate the regulation of target mRNAs.

Broadening the Definition of Bacterial Small RNAs: Characteristics and Mechanisms of Action

The first report of trans-acting RNA-based regulation in bacterial cells dates back to 1984. Subsequent studies in diverse bacteria unraveled shared properties of trans-acting small regulatory RNAs, forming a clear definition of these molecules. These shared characteristics have been used extensively to identify new small RNAs (sRNAs) and their interactomes. Recently however, emerging technologies able to resolve RNA-RNA interactions have identified new types of regulatory RNAs. In this review, we present a broader definition of trans-acting sRNA regulators and discuss their newly discovered intrinsic characteristics.