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.     

Competition among Hfq-binding small RNAs in Escherichia coli

A major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.     

Characterization of the small RNA transcriptome in plant–microbe (Brassica/Erwinia) interactions by high-throughput sequencing

Non-coding, small RNAs (sRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans; however, the role and mechanisms of these sRNA in plant immunity is largely unknown. To determine possible roles of sRNA in plant–pathogen interaction, we carried out a high-throughput sRNA sequencing of Brassica campestris using non-infected plants and plants infected with Erwinia carotovora. Consistent with our hypothesis that distinct classes of host sRNAs alerts their expression levels in response to infection, we found that: (1) host 28-nt sRNAs were strongly increased under pathogen infection; and (2) a group of host sRNAs homologous to the pathogen genome also accumulated at significantly higher level. Our data thus suggest several distinct classes of the host sRNAs may enhance their function by up-regulation of their expression/stability in response to bacterial pathogen challenges.     

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。     

Molecular call and response: the physiology of bacterial small RNAs

The vital role of bacterial small RNAs (sRNAs) in cellular regulation is now well-established. Although many diverse mechanisms by which sRNAs bring about changes in gene expression have been thoroughly described, comparatively less is known about their biological roles and effects on cell physiology. Nevertheless, for some sRNAs, insight has been gained into the intricate regulatory interplay that is required to sense external environmental and internal metabolic cues and turn them into physiological outcomes. Here, we review examples of regulation by selected sRNAs, emphasizing signals and regulators required for sRNA expression, sRNA regulatory targets, and the resulting consequences for the cell. We highlight sRNAs involved in regulation of the processes of iron homeostasis (RyhB, PrrF, and FsrA) and carbon metabolism (Spot 42, CyaR, and SgrS).     

A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.     

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。     

细菌sRNA基因及其靶标预测研究进展

摘要：细菌sRNA是一类长度在40～500 nt之间的非编码RNA,主要以不完全碱基配对方式与靶标mRNA5′端相互作用进而发挥其生物学功能。鉴于预测方法可以为细菌sRNA及其靶标的实验发现提供指导,因此,细菌sRNA与靶标预测研究受到了广泛重视。文章首先将sRNA预测方法分为3类,分别是基于比较基因组学的预测方法、基于转录单元的预测方法和基于机器学习的预测方法;其次,将sRNA靶标预测方法分为2类,分别是序列比较方法与基于RNA二级结构的预测方法;最后对各类方法的原理、核心思想、优点和局限性进行了分析,并探讨了进一步的发展方向。     

Identification of bacterial sRNA regulatory targets using ribosome profiling

Bacteria express large numbers of non-coding, regulatory RNAs known as ‘small RNAs’ (sRNAs). sRNAs typically regulate expression of multiple target messenger RNAs (mRNAs) through base-pairing interactions. sRNA:mRNA base-pairing often results in altered mRNA stability and/or altered translation initiation. Computational identification of sRNA targets is challenging due to the requirement for only short regions of base-pairing that can accommodate mismatches. Experimental approaches have been applied to identify sRNA targets on a genomic scale, but these focus only on those targets regulated at the level of mRNA stability. Here, we utilize ribosome profiling (Ribo-seq) to experimentally identify regulatory targets of the Escherichia coli sRNA RyhB. We not only validate a majority of known RyhB targets using the Ribo-seq approach, but also discover many novel ones. We further confirm regulation of a selection of known and novel targets using targeted reporter assays. By mutating nucleotides in the mRNA of a newly discovered target, we demonstrate direct regulation of this target by RyhB. Moreover, we show that Ribo-seq distinguishes between mRNAs regulated at the level of RNA stability and those regulated at the level of translation. Thus, Ribo-seq represents a powerful approach for genome-scale identification of sRNA targets.