细菌中非编码小RNA的筛选及鉴定

细菌非编码小RNA(small non-coding RNA,sRNA)是一类长度在50-200个核苷酸,不编码蛋白质的RNA.它们通过碱基配对识别靶标mRNA,在转录后水平调节基因的表达,是细菌代谢、毒力和适应环境压力的重要调节因子.近年来,随着生物信息学和RNA组学技术应用于细菌sRNA的筛选,sRNA已被证实存在于大肠埃希杆菌(Escherichia coli),铜绿假单胞菌(Pseudomonas aeruginosa)、霍乱弧菌(Vibrio cholerae)等细菌中,是细菌基因调控中新的调节因子.本文对细菌中非编码小RNA的筛选和鉴定技术作一个简要论述.     

细菌中的小RNA

小RNA（sRNA）或非编码RNA（ncRNA）在原核生物和真核生物中广泛分布。迄今,在各种细菌中共发现超过150种sRNA,在大肠杆菌中发现了约80种sRNA。sRNA通过与靶mRNA配对而发生作用,导致mRNA翻译和稳定性的变化;sRNA的功能涉及从结构调节到催化作用,影响生物体内各种各样的加工过程,一个单独的sRNA就能调控大量的基因并对细胞生理产生深远影响。目前,对sRNA的研究主要采用生物信息学预测结合分子生物学实验的方法。     

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。     

细菌非编码小RNA功能及作用机制研究进展

生物体中除了编码蛋白质的mRNA外,还存在多种具有重要调控功能的非编码RNA。细菌中长度50~500 nt的非编码RNA通常定义为sRNA。sRNA在细菌的整个生命活动中发挥着极为广泛的作用,在感受环境压力、基因表达、细胞周期乃至个体发育等过程中均具有重要的调控作用。sRNA的功能学和调控机制的研究已成为当今细菌学研究的热点。本研究就细菌中的sRNA的特征,在细菌中的作用和作用机制进行文献综述。     

细菌sRNA对环境胁迫的响应机制

细菌小RNA (Small RNAs,sRNAs)是一类长度大约在40?400个核酸之间,不编码蛋白质的RNA,在细菌适应环境方面起重要的调节作用。当环境中温度、营养、外膜蛋白、pH、铁等条件改变时,sRNA常常通过连接双组分信号转导系统和调节蛋白,来传递压力信号并调节应激响应,其作用方式一般是通过碱基互补配对的方式与靶mRNA结合,从而调控靶mRNA的翻译和稳定性;或直接与靶标蛋白质结合,调节靶标蛋白质的生物活性。本文总结了细菌在多种环境压力下,sRNA的调控响应机制。     

基于转录组测序探索创伤弧菌MO6-24/O小RNA

目的:基于转录组测序注释,探索创伤弧菌MO6-24/O小RNA(sRNA)。方法:在海洋培养基2216E中培养创伤弧菌MO6-24/O,收集从对数生长期到静止期不同时间点的细菌,提取细菌RNA,通过富集sRNA以及去除核糖体RNA进行建库和测序,然后将读段匹配到创伤弧菌MO6-24/O基因组并进行注释,最后进行验证。结果:发现59个顺式编码sRNA、102个反式编码sRNA,并进行了部分验证。结论:鉴定出创伤弧菌MO6-24/OsRNA。     

基于转录终点序列特征预测大肠杆菌sRNA

细菌sRNA是一类长度在40～500nt的调控RNA,在细菌与环境相互作用中发挥重要功能,因此,细菌sRNA识别研究具有重要意义。然而,与蛋白编码基因具有易于识别的特征不同,目前细菌sRNA识别仍是一件比较困难的事。此方法介绍了一个基于已知细菌sRNA转录终点的碱基频率矩阵来识别sRNA的预测策略,并在大肠杆菌K-12 MG1655中进行了sRNA的预测。结果表明,该模型在独立测试集中具有较高的特异性和阳性检出率,因此,这一方法将为实验发现细菌sRNA提供较好的生物信息学支持。     

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。     

非编码小RNA对细菌毒力及耐药性的调控作用研究进展

近年来的研究发现,细菌非编码小RNA (small non-coding RNA, sRNA)对其不同生理进程起到了重要的调控作用。随着大量sRNA被发现并鉴定,细菌sRNA的功能被逐步阐明,其可在转录后水平广泛调控细菌的生理代谢、毒力及耐药性等。本文综述了sRNA对细菌毒力和耐药性调控作用的研究进展,对揭示细菌转录后水平毒力及耐药性调控机制具有一定意义。     

细菌小RNA识别研究方法进展

细菌小RNA(sRNA)是一类长度在50～500 nt的调控RNA,主要通过与靶标mRNA以不完全配对方式结合来发挥调控作用,sRNA参与细菌基因的转录、翻译、mRNA稳定、成熟和加工等多个过程,因此,sRNA的发现及功能注释对了解细菌的致病机制至关重要。该文将对研究sRNA的各种计算机和实验方法进行综述,阐述各种方法的优缺点,并讨论今后的发展方向。     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.     

A small RNA targets pokeweed antiviral protein transcript

Ribosome‐inactivating proteins (RIPs) are a class of plant defense proteins with N‐glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad‐spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.     

原核生物中小RNA的功能及研究进展

在原核生物基因组中,除了tRNA、rRNA和mRNA这3种我们很熟悉的RNA以外,目前已经知道还含有许多编码非常规调控的RNA。这些RNA的长度为50～400核苷酸,通常由原核生物的基因间区编码,但并不翻译成蛋白质,因此被称为非编码小RNA（sRNA）,简称小RNA或非编码RNA。近年来,随着生物信息学和分子生物学技术的不断发展,在原核生物体内陆续发现了上百种sRNA分子,这些sRNA分子具有多样的生物学功能,作为一类新发现的基因表达调控子已经引起了高度的重视。     

Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs

Many classes of small RNA (sRNA) involved in RNA silencing are generated by double-stranded RNA (dsRNA) processing. Although principles of sRNA biogenesis have emerged, newly identified classes of sRNAs have features that suggest additional biogenesis mechanisms. Tetrahymena thermophila expresses one such class, comprising sRNAs of 23 and 24 nucleotides (nt) with an absolute strand bias in accumulation. Here we demonstrate sRNA production by the T. thermophila Dicer Dcr2 and the RNA-dependent RNA polymerase Rdr1, which purifies as a multisubunit RNA-dependent RNA polymerase complex (RDRC). Dcr2 and RDRC interact, stimulating Dcr2 activity. Moreover, Dcr2 specificity is influenced by RDRC beyond this physical interaction, as Dcr2 generates discrete 23- and 24-nt sRNAs only from dsRNA with a 5'-triphosphate. These findings suggest that sRNA strand bias arises from Dcr2 processing polarity, conferred by physical and functional coupling of RDRC and Dicer enzymes.     

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.     

Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA‐BINDING PROTEIN 1–ribonucleoprotein complex

In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense‐related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL‐RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem‐localized protein kinase, PSRPK1. During long‐distance transport, PSRP1–sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1–sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants.     

Systematic Analysis of Small RNAs Associated with Human Mitochondria by Deep Sequencing: Detailed Analysis of Mitochondrial Associated miRNA

Mitochondria are one of the central regulators of many cellular processes beyond its well established role in energy metabolism. The inter-organellar crosstalk is critical for the optimal function of mitochondria. Many nuclear encoded proteins and RNA are imported to mitochondria. The translocation of small RNA (sRNA) including miRNA to mitochondria and other sub-cellular organelle is still not clear. We characterized here sRNA including miRNA associated with human mitochondria by cellular fractionation and deep sequencing approach. Mitochondria were purified from HEK293 and HeLa cells for RNA isolation. The sRNA library was generated and sequenced using Illumina system. The analysis showed the presence of unique population of sRNA associated with mitochondria including miRNA. Putative novel miRNAs were characterized from unannotated sRNA sequences. The study showed the association of 428 known, 196 putative novel miRNAs to mitochondria of HEK293 and 327 known, 13 putative novel miRNAs to mitochondria of HeLa cells. The alignment of sRNA to mitochondrial genome was also studied. The targets were analyzed using DAVID to classify them in unique networks using GO and KEGG tools. Analysis of identified targets showed that miRNA associated with mitochondria regulates critical cellular processes like RNA turnover, apoptosis, cell cycle and nucleotide metabolism. The six miRNAs (counts >1000) associated with mitochondria of both HEK293 and HeLa were validated by RT-qPCR. To our knowledge, this is the first systematic study demonstrating the associations of sRNA including miRNA with mitochondria that may regulate site-specific turnover of target mRNA important for mitochondrial related functions.