Dynamics of Salmonella small RNA expression in non-growing bacteria located inside eukaryotic cells

Small non-coding regulatory RNAs (sRNAs) have been studied in many bacterial pathogens during infection. However, few studies have focused on how intracellular pathogens modulate sRNA expression inside eukaryotic cells. Here, we monitored expression of all known sRNAs of Salmonella enterica serovar Typhimurium (S. Typhimurium) in bacteria located inside fibroblasts, a host cell type in which this pathogen restrains growth. sRNA sequences known in S. Typhimurium and Escherichia coli were searched in the genome of S. Typhimurium virulent strain SL1344, the subject of this study. Expression of 84 distinct sRNAs was compared in extra- and intracellular bacteria. Non-proliferating intracellular bacteria upregulated six sRNAs, including IsrA, IsrG, IstR-2, RyhB-1, RyhB-2 and RseX while repressed the expression of the sRNAs DsrA, GlmZ, IsrH-1, IsrI, SraL, SroC, SsrS(6S) and RydC. Interestingly, IsrH-1 was previously reported as an sRNA induced by S. Typhimurium inside macrophages. Kinetic analyses unraveled changing expression patterns for some sRNAs along the infection. InvR and T44 expression dropped after an initial induction phase while IstR-2 was induced exclusively at late infection times (> 6 h). Studies focused on the Salmonella-specific sRNA RyhB-2 revealed that intracellular bacteria use this sRNA to regulate negatively YeaQ, a cis-encoded protein of unknown function. RyhB-2, together with RyhB-1, contributes to attenuate intracellular bacterial growth. To our knowledge, these data represent the first comprehensive study of S. Typhimurium sRNA expression in intracellular bacteria and provide the first insights into sRNAs that may direct pathogen adaptation to a non-proliferative state inside the host cell.     

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.     

植物小分子RNA研究进展

植物体内存在多种不同类型的小分子RNA(small RNA,sRNA),在调节植物生长发育、抑制转座子活性和抵御逆境等过程中发挥着重要的作用。近年来,人们在sRNA的产生机制、效应复合物的形成和对靶基因的调控方式及其生物学功能等方面的研究取得了很大进展。该文对这些进展作简要介绍。     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.     

Bacterial Small Regulatory RNAs and Hfq Protein

Small regulatory RNA (sRNA) is a unique noncoding RNA involved in regulation of gene expression in both eukaryotic and bacterial cells. This short review discusses examples of positive and negative translation regulation by sRNAs in bacteria and participation of Hfq in these processes. The importance of structure investigation of nucleotide–protein and RNA–protein complexes for designing a model of Hfq interaction with both mRNA and sRNA simultaneously is demonstrated.     

病原菌非编码sRNA的功能及研究新方法

细菌中的非编码小RNA（small RNA, sRNA）作为一种靶向调控分子在细胞生理代谢过程中具有重要作用。sRNA作用于特定靶标,调控基因的表达。大肠杆菌大约有100种sRNA,其中1/3 sRNA需要伴侣蛋白Hfq的介导。病原细菌中sRNA分子如何调控致病基因的表达,目前研究仍处于初级阶段。本文将从生物膜形成、细菌耐药性以及对宿主的影响等方面,结合新颖的sRNA的研究方法,综述sRNA在调控代谢网络及控制病原菌致病性方面的作用。     

Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing

Quorum sensing is a mechanism of cell‐to‐cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell‐population density. At the core of the Vibrio cholerae quorum‐sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named the quorum regulatory RNAs 1–4 (Qrr1–4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild‐type quorum‐sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA‐activator LuxO and one involving the sRNA‐target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1–4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum‐sensing‐regulated target genes.     

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.