细菌非编码小RNA的筛选及鉴定方法

细菌非编码小RNA（smallnon．codingRNAs,sRNAs）是一类长度为50～500nt、不编码蛋白质的功能RNA,在应对胁迫、毒力产生和新陈代谢等生命过程中起重要的调控作用。其主要通过碱基配对与靶mRNA发生作用,导致mRNA翻译和稳定性改变,从而在转录后水平调节基因的表达,最终影响细菌各种生命活动。近年来,利用生物信息学和分子生物学技术,已在细菌中筛选并鉴定得到了几百个sRNA。该文对细菌sRNA的筛选和鉴定方法作一简要综述。     

革兰氏阳性菌非编码小RNA的研究进展

sRNA（非编码小RNA）通过碱基配对的方式与靶mRNA结合,抑制或激活转录过程、调节蛋白质的表达,以核酸的形式发挥其生物学功能。随着RNA深度测序（RNAseq）技术、生物信息学预测以及实验分析手段的日渐发展和完善,数以百计的sRNA被发现并得到验证。作为转录后调控因子,sRNA因在诸多生理过程中起到了关键的调节作用而得到了广泛的关注。以革兰氏阳性菌为切入点,总结了近年来sRNA的筛选、鉴定和功能研究等方面取得的进展,梳理分析了sRNA调控与毒力因子、群体感应、铁代谢和双组分系统等之间的内在联系,并展望了sRNA未来的研究方向。     

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.     

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.     

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.     

Paradoxical suppression of small RNA activity at high Hfq concentrations due to random-order binding

Small RNAs (sRNAs) are important regulators of gene expression during bacterial stress and pathogenesis. sRNAs act by forming duplexes with mRNAs to alter their translation and degradation. In some bacteria, duplex formation is mediated by the Hfq protein, which can bind the sRNA and mRNA in each pair in a random order. Here we investigate the consequences of this random-order binding and experimentally demonstrate that it can counterintuitively cause high Hfq concentrations to suppress rather than promote sRNA activity in Escherichia coli. As a result, maximum sRNA activity occurs when the Hfq concentration is neither too low nor too high relative to the sRNA and mRNA concentrations (‘Hfq set-point’). We further show with models and experiments that random-order binding combined with the formation of a dead-end mRNA–Hfq complex causes high concentrations of an mRNA to inhibit its own duplex formation by sequestering Hfq. In such cases, maximum sRNA activity requires an optimal mRNA concentration (‘mRNA set-point’) as well as an optimal Hfq concentration. The Hfq and mRNA set-points generate novel regulatory properties that can be harnessed by native and synthetic gene circuits to provide greater control over sRNA activity, generate non-monotonic responses and enhance the robustness of expression.     

Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base‐pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base‐pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO‐domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO‐domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO‐domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.     

A Peroxide-Responding sRNA Evolved from a Peroxidase mRNA

Small RNAs (sRNAs) are important gene regulators in bacteria, but it is unclear how new sRNAs originate and become part of regulatory networks that coordinate bacterial response to environmental stimuli. Using a covariance modeling-based approach, we analyzed the presence of hundreds of sRNAs in more than a thousand genomes across Enterobacterales, a bacterial order with a confluence of factors that allows robust genome-scale sRNA analyses: several well-studied organisms with fairly conserved genome structures, an established phylogeny, and substantial nucleotide diversity within a narrow evolutionary space. We discovered that a majority of sRNAs arose recently, and uncovered protein-coding genes as a potential source from which new sRNAs arise. A detailed investigation of the emergence of OxyS, a peroxide-responding sRNA, revealed that it evolved from a fragment of a peroxidase messenger RNA. Importantly, although it replaced the ancestral peroxidase, OxyS continues to be part of the ancestral peroxide-response regulon, indicating that an sRNA that arises from a protein-coding gene would inherently be part of the parental protein’s regulatory network. This new insight provides a fresh framework for understanding sRNA origin and regulatory integration in bacteria.     

Conserved arginines on the rim of Hfq catalyze base pair formation and exchange

The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq’s chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq’s chaperone function.     

小RNA对胞内菌生长、毒力及铁水平的调节作用

细菌小RNA(small RNA,sRNA)是一类长度为50~500个碱基,具有调控转录、翻译和mRNA稳定性的非编码调节性RNA。随着越来越多的sRNA被鉴定,部分细菌的sRNA功能已逐步阐明,主要参与调控细菌的基因表达、增殖、毒力及对环境的应激反应等生物学过程。本文就胞内菌(如沙门菌、李斯特菌、嗜肺军团菌等)sRNA对其自身在宿主细胞内的生长、毒力和铁水平的调控作用进行综述。