tRNA来源的小片段RNA——新的基因表达调控因子

转移核糖核酸(tRNA)是蛋白质合成的关键接头分子,特异性识别信使RNA(mRNA)的密码子信息,将其接载的氨基酸基团掺入到新生多肽链中。最新研究表明,在很多物种中,在某些特定情况下,tRNA或其前体被特异性剪切产生tRNA来源的小片段RNA(tRNA-derived fragment,tRF)。这类tRF是一类新的基因表达调控因子,其发挥作用的机制多样,如某些tRF以microRNA方式抑制mRNA翻译;某些tRF作为逆转录病毒RNA基因组的逆转录引物;而某些tRF参与了前体rRNA剪切复合物的组装。此外,细胞受胁迫产生的带有多聚鸟苷酸模块的tRF则会竞争性抑制延伸因子elF4G与mRNA的结合,从而抑制蛋白质翻译。随着研究的继续深入,对tRF的发生发展、作用机制以及在疾病中的潜在作用将会进一步丰富。拟从tRF作为新的基因表达调控分子的角度,简要介绍tRF发挥作用的分子机制。     

tRNA衍生片段的鉴定及对靶分子的调节机制

随着测序技术的发展和对tRNA衍生小分子(tRNA-derived small RNA,tsRNAs)的深入研究,越来越多的tsRNAs及其功能在各物种中被鉴定。tsRNAs根据切割位点的不同可分为tRNA衍生片段(tRNA-derived fragment,tRF)和tRNA应激诱导RNA(tRNA-derived stress-induced RNA,tiRNA),其中tRF是一类具有调节功能的非编码RNA。为了加深对tRF的研究,近年来一些基于测序数据的tRF鉴定方法和相关数据库不断涌现,前者主要包括Telonis等人的算法和tDRmapper方法,后者主要有tRFdb、tRF2Cancer和MINTbase等。同时这两者为tRF的深入研究提供了更有效的工具。大量的研究表明,tRF主要以类似miRNA的方式对RNA、DNA及蛋白质进行调节,但也存在特异的作用方式。随着对这三者的深入研究,研究人员发现tRF在人类疾病的各种生物过程中也扮演着重要的角色,例如可以作为生物标志物。因此本文主要对tRF的鉴定方法、数据库、对靶分子的调节机制及其与人类疾病的关系作一综述。     

水稻雄配子体发育过程中tRNA片段的生物信息学分析

tRNA片段(tRF)是tRNA通过非随机剪切产生的RNA片段, 其产生和功能机制尚不明确; 而在水稻(Oryza sativa)雄配子体发育过程中, 人们对tRNA更是知之甚少。通过高通量测序, 在水稻雄配子体发育过程中发现了长度范围较大的tRFs; 进一步采用logo对tRFs两端的序列进行分析, 发现了4个有序列特征(其中3个未见报道)和1个无序列特征的酶切位点; 通过NCBI Blast预测了tRF靶基因, 发现其大多靶向转座因子。研究结果对揭示tRF产生机制以及水稻雄配子体发育研究有一定的参考价值。     

转移核糖核酸(tRNA)与癌症

转移核糖核酸(transfer RNA, tRNA)在蛋白质生物合成过程中起关键作用,是将遗传信息翻译成蛋白质一级结构的接头分子。tRNA长久以来一直被认为是基因表达调控过程中的执行者而不具备调控功能,更不曾与癌症的发生联系起来。最新研究表明,某些tRNA在癌细胞中异常表达,与癌症的发生和发展有密切联系。tRNA来源的小分子非编码RNA (tRFs和tiRNAs)是一类新的基因表达调控分子,tRFs可以调控癌基因的表达或者与RNA结合蛋白相互作用来调控癌细胞增殖和细胞周期进程。tRNA的转录后修饰能够调控mRNA翻译过程,进而影响癌细胞的生长。随着测序技术的发展,tRNA在癌症发生和发展中的调控作用成为近年来的研究热点,现将从"tRNA分子调控癌症的发生和发展"、"tRNA来源的小分子非编码RNA与癌症"以及"tRNA修饰与癌症"三个方面综述tRNA分子在癌症发生和发展中的调控功能。     

来源于tRNA的小分子RNA——降解碎片还是新的调控分子

具有经典三叶草结构的tRNA作为细胞蛋白质合成机器的重要元件,已经拥有几十年深入细致的研究历史.但是,对于其功能的认识远没有止境,尤其在其作为潜在的基因表达调控分子前体的功能目前正逐渐被人们认识.最新的多项研究结果表明,在多种细胞系中通过高通量测序发现某种来源于tRNA的小片段RNA,这些剪切产物被认为与多种microRNA加工体系关键分子(如Dicer、Ago家族中的蛋白质)具有相互作用的能力.同时,报告基因检测系统的研究结果也暗示,这些小片段RNA具有类似microRNA的潜在调控功能,可能在细胞应对外界环境刺激时发挥重要的调节作用.如其具体的作用机制能够被更多的实验结果阐明,将极大地扩展我们对于非编码RNA调控功能的认识.     

tRNA衍生片段和tRNA半分子的生物学功能及其在疾病发生中的作用

tRNA衍生片段(tRNA-derived RNA fragment,t RF)和tRNA半分子(tRNA halves,ti RNA)由成熟tRNA或其前体tRNA在不同位点特异性剪切产生,它们是一类广泛存在于原核生物和真核生物转录组中的非编码小RNA分子.t RF主要有tRF-5、tRF-3和tRF-1等3亚类,分别来自成熟tRNA的D环至反密码环茎区间切割至5′端、T环开始至3′端和前体tRNA的3′端尾部,其长度为14~30个核苷酸(nucleotide,nt).ti RNA主要有5′ti RNA和3′ti RNA等2亚类,是在成熟tRNA反密码子环处切割分别产生,其长度为29~50 nt.t RF和ti RNA具有多种生物学功能,既可以在应激反应中作为信号分子,又可以作为基因表达的调节者.它们与人类多种疾病(如肿瘤、神经退行性疾病、代谢性疾病和传染病等)的发生密切相关,有希望成为疾病诊断的新型标志物.本文就t RF和ti RNA的分类、生物学功能以及与人类疾病的关系作一综述.     

血管生成素与tRNA片段化

tRNA主要功能是转运氨基酸参与蛋白质合成,在蛋白质生物合成过程中起着关键性的作用．近年来发现,tRNA是细胞内小RNA分子的重要来源,具有其它重要的生物学功能．来源于成熟tRNA分子的tRNA片段根据切割位置及生成机制的不同,主要分为两类：一类是tRNA半分子（tRNA halves）;另一类是较小的tRNA片段,称为tRFs( tRNA fragments)．在哺乳动物细胞中,tRNA半分子由血管生成素在tRNA分子反密码环处切割生成．本文主要针对tRNA半分子的加工机制、功能及在临床上的潜在应用进行综述．     

氨酰-tRNA合成酶对tRNA的识别

氨酰-tRNA合成酶(aaRS)与tRNA的相互作用保证了蛋白质生物合成的忠实性. 氨酰-tRNA合成酶对tRNA识别的专一性依赖于aaRS特定的催化结构域和tRNA分子特异的三级结构构象. 反密码子和接受茎(包括73位)在大多数aaRS对tRNA分子的识别过程中起着关键作用, 其他部位如可变口袋、可变(茎)环等, 甚至修饰核苷酸对于一些识别过程也有重要作用.     

十字花科黑腐病菌中一个与抗逆有关的小RNA的鉴定

十字花科黑腐病菌(Xanthomonas campestris pathovar campestris,Xcc),是引起十字花科植物黑腐病的病原菌。Xcc要经历寄生、腐生等多种环境变化,为适应这些环境变化,需要调控相应基因的表达。除蛋白外,小RNA在基因表达调控中也起到关键作用。本实验室前期实验从Xcc 8004中鉴定出数百个小RNA,但是绝大多数小RNA的功能仍然未知。本研究通过构建一个小RNA(sRNA3843)的过量表达株来研究其生物学功能。确定该小RNA过量表达后,对其过量表达株OE3843进行了一系列的表型检测。结果发现,s RNA3843过量表达株OE3843对金属离子Cu^2+、Zn^2+、Cd^2+及蛋白变性剂SDS的耐受能力明显下降,表明s RNA3843与Xcc的抗逆有关。本研究的实验结果为深入研究小RNA在Xcc抗逆中所起的作用及其作用机理打下基础。     

十字花科黑腐病菌群体密度和DSF信号因子负调控Ⅲ型分泌系统

十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)是能在全世界引起十字花科植物黑腐病的革兰氏阴性细菌。群体感应(quorum sensing,QS)是细菌通过感应菌群生长密度调控自身生理活动和致病因子的作用。Xcc QS信号分子为DSF(diffusible signal factor)因子,感受系统由rpf(regulation of pathogenicity factors)基因簇编码。Ⅲ型分泌系统(T3SS)是Xcc与寄主植物相互作用并致病的关键系统,Xcc的Ⅲ型分泌系统是由hrp(hypersensitive response and pathogenicity)基因簇所编码的。研究rpf/DSF系统对Ⅲ型分泌系统的调控作用,对于揭示该病原菌致病分子机制,以及植物病害防治具有重要的意义。针对Xcc 8004菌株,利用连有hrp B基因启动子区的p LGUS报告质导入Xcc 8004中,在XCM诱导培养基中添加DSF因子后通过测定GUS酶活对比DSF对hrp基因的调控作用。结果发现,随着菌体浓度的增长,hrp B基因的表达呈下降趋势;额外加入DSF因子后hrp B基因的表达量呈现下降趋势。十字花科黑腐病菌中菌体密度和信号扩散因子对Ⅲ型分泌系统有负调控作用。     

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.     

Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

十字花科黑腐病菌hrpG基因原核表达

在十字花科黑腐病菌(Xcc)中,hrp基因对寄主的致病性和非寄主的超敏反应中起核心作用,而hrpG对整个hrp基因簇起调控作用.HrpG为OmpR家族的双组分系统感受调控蛋白,含有两个结构域,分别是N端Response_reg和C端Trans reg_C.本研究利用表达载体pQE-30 Xa,成功构建了HrpG的表达重组子,在E.coli M15 [pREP4]中进行诱导表达.通过调节诱导温度、IPTG浓度和诱导时间最终确定在温度为20℃,IPTG浓度为0.8 mmol/L,诱导表达4 h.hrpG基因在宿主细胞E.coli M15获得高效可溶性表达.目前尚未有可溶性HrpG蛋白获得成功表达的报导,本研究中获得HrpG蛋白在大肠杆菌获得大量可溶性的表达,将为in vitro研究HrpG的生理活性,特异的结合位点和调控功能研究打下良好基础.     

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.     

Light signaling mediated by PAS domain-containing proteins in Xanthomonas campestris pv. campestris

Per-ARNT-Sim (PAS) domains are important signalling modules that possibly monitor changes in various stimuli such as light. For the majority of PAS domains that have been identified by sequence similarity, the biological function of the signalling pathways has not yet been experimentally investigated.Thirty-three PAS proteins were discovered in Xanthomonas campestris pv. campestris(Xcc) by genome/proteome analysis. Thirteen PAS proteins were identified as contributing to light signalling and Xcc growth, motility or virulence using molecular genetics and bioinformatics methods. The PAS domains played important roles in light signalling to regulate the growth, motility and virulence of Xcc. They might be regulated by not only light quality (wavelength)but also quantity (intensity) as potential light-signalling components. Evaluating the light wavelength, three light-signalling types of PAS proteins in Xcc were shown to be involved in blue light signalling, tricolour (blue, red and far red)signalling or red/far-red signalling. This showed that Xcc had evolved a complicated light-signalling system to adapt to a complex environment.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.