原核细胞microRNA特性及其作用机制研究进展

张新蔚，黄燕颖，严 杰，孙爱华



原核细胞microRNA特性及其作用机制研究进展

张新蔚1,3，黄燕颖1,3，严 杰2，孙爱华3

microRNA为一类非编码小RNA，主要通过其种子序列(seed sequence，SS)与靶mRNA位于5′端的SS结合序列特异性结合后抑制靶mRNA转录或降解靶mRNA，从而在转录后水平负调控靶基因表达。microRNA首先发现于秀丽隐杆线虫(Caenorhabditiselegans)，此后发现各种真核细胞均存在大量各种microRNA。真核细胞首先转录出microRNA前体，经剪切后成为21～23 nt有功能的microRNA，大多与靶mRNA的3′端序列结合后引起靶mRNA翻译抑制或降解。近年不少细菌等原核细胞microRNA被发现，但原核细胞microRNA不需剪切即有活性，大小为50～400 nt，其特性、作用位点和机制等也与真核细胞有一定差异。本文简要介绍真核和原核细胞基因表达调控主要机制、microRNA特点及其调控基因表达的机制，以期为深入研究原核细胞型病原微生物基因表达调控与致病机制奠定基础。

原核细胞；基因表达；调控；microRNA；作用机制

Supported by the National Natural Science Foundation of China (No. 81271893) and the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents (No. 2012-241) Corresponding author: Sun Ai-hua,Email: sunah123@126.com

生物大分子有蛋白、多糖、核酸及其复合物，在生命体生理或病理过程中发挥关键作用。基因是DNA分子中携带遗传信息的片段，其表达产物为蛋白或多肽。酶是一类具有催化功能的蛋白，上述生物大分子合成与降解均依赖于酶的作用。因此，了解基因表达调控的物质基础及其工作机制具有重要意义。基因表达过程分为mRNA转录和翻译(转录后)两大环节。转录因子(transcription factor，TF)是能与DNA结合并在转录水平上靶基因表达的蛋白，但近年发现一类称之为microRNA的非编码小RNA也能在转录后水平上调控靶基因表达。本文简要介绍真核或原核细胞TF尤其是microRNA调控靶基因表达的主要机制及其差异。

1 TF调控靶基因表达的主要机制

一般认为，真核或原核细胞基因表达调控主要依赖于TF，microRNA通常仅有微调作用。一个基因编码的蛋白对该基因或另一基因表达的调节作用分别称为顺式或反式调节作用，包括基因转录的开启和关闭。

1.1 真核细胞TF调控靶基因表达机制[1-7]在真核细胞中，顺式作用元件(cis-acting element)和反式作用因子(trans-acting factor)及其相互作用是转录水平调控基因表达的基本机制。顺式作用元件为位于基因旁侧、可调控或影响该基因表达的特殊序列，根据功能不同可分为启动子(promotor)、增强子(enhanser)、沉默子(silencer)应答元件(response element)。反式作用因子能直接或间接识别并结合另一基因顺式作用元件核心序列、调控该基因转录水平或过程，以TF最为重要和常见。TF特异性识别靶基因启动子区中TF结合位点(TF-binding site，TFBS)并与之结合，激活或抑制基因转录。多个TFBS可组成顺式调节模块(cis-regulatory modules，CRM)，大多数基因表达由多种TF通过CRM调控，一个TF也可通过不同的CRM调控多个基因表达。根据功能不同，TF可分为转录激活子(transcription activator)和后者称转录抑制子(transcription inhibitor)，前者为增强子结合蛋白(enhancer-binding protein，EBP)，与增强子序列结合后上调基因表达，后者属于沉默子结合蛋白(silencer-binding protein，SBP)，与沉默子序列结合后下调基因表达。增强子和沉默子序列位于转录起始点一定距离外(1-30 kb)，其作用不受序列方向的影响，也可对异源基因发挥作用。此外，TF对某一靶基因表现为表达上调作用，但对另一靶基因可呈现为表达抑制作用。

1.2 原核细胞基因表达调控主要机制[6-11]大多数原核细胞通过操纵子调控基因表达。操纵子由结构基因和调控序列组成，前者常为功能上有关联、串联排列的数个基因，共同构成编码区，后者包括启动子、操纵元件(operator)以及一定距离外的调节基因。操纵元件是一段能被特异的阻遏蛋白(repressor)识别并结合的DNA序列，与启动子序列毗邻甚至重叠。阻遏蛋白与操纵子结合后，阻断RNA聚合酶与启动子结合或阻碍RNA聚合酶沿DNA向前移动，在转录水平上抑制基因表达。许多原核细胞基因操纵子中还有一段独特的DNA序列，与激活蛋白(activator)结合后增强RNA聚合酶活性，在转录水平上调基因表达。调节基因编码能与操纵序列结合的调控蛋白(modulin)，可分为特异因子、阻遏蛋白和激活蛋白三类，其中特异因子决定RNA聚合酶对一个或一套启动子序列的特异性识别和结合能力。

任何生物体必然与其生存环境发生相互联系并受之影响，由信号传导系统(signaling system)感受外界信号并作出应答，其中跨膜感受器(sensor)蛋白激酶接受环境分子信号并向胞内传递，最终通过TF调控相关基因表达来应对环境变化。与真核细胞比较，细菌等原核细胞信号传导系统相对简单，一般由两类分工明确的蛋白组成，故称之二元信号传导系统(two-component signaling system，TCSS)：①跨膜传感器蛋白(sensor protein，SP)：多为组氨酸激酶，少数为丝氨酸/苏氨酸激酶；②胞浆内应答调节蛋白(response regulator protein，RRP)，通常被跨膜传感激酶磷酸化后激活，具有类似真核细胞TF功能，通过调节靶基因表达水平对环境信号进行适应性应答。例如，金黄色葡萄球菌(Staphylococcusaureus)ArlRS是调控许多毒力基因表达的TCSS，ArlS为SP、ArlR为RRP，ArlR上调sdrC/D/rE、tcaB和ssaA等毒力基因表达，但下调lukD/E、phlC和hlgC毒素基因表达；大肠埃希菌(Escherichiacoli)EnvZ/OmpR 是环境渗透压TCSS，EnvZ为SP、OmpR为RRP，OmpR可分别与外膜孔蛋白OmpF和OmpC编码基因启动子区结合，低渗时OmpF表达上调、OmpC表达下调，高渗时OmpF和OmpC表达水平相反。

2 真核或原核细胞microRNA产生及特性

microRNA首先发现于秀丽隐杆线虫(Caenorhabditiselegans)，此后发现真核或原核细胞均存在大量各种microRNA[12-13]。真核或原核细胞microRNA产生过程、分子大小与结构以及作用机制均存在一定差异。

2.1 真核细胞microRNA产生及特性[14-16]在RNA聚合酶的作用下，真核细胞microRNA编码基因转录产生pri-microRNA，随后Drosha 酶将pri-microRNA剪切成pre-microRNA，然后Ran-GTP 和 Exportin-5蛋白将pre-microRNA从细胞核运送到细胞质，再经胞浆Dicer核酸酶剪切成通常为21～23 nt左右成熟的单链小RNA(microRNA)。胞浆内RNA诱导沉默复合体(RNA-induce silencing complex，RISC)可识别microRNA并将其递呈至靶mRNA，大多数microRNA与靶mRNA的3’端非翻译区(untranslated region，UTR)序列互补，抑制该mRNA翻译或引起mRNA降解。动物约有1/3基因受到microRNA调控。

2.2 原核细胞microRNA产生及特性[17-20]原核细胞microRNA长度为50～400 nt，转录后一般不经过加工即有活性。原核细胞microRNA起始一端折叠成茎环结构，转录终止于一个Rho不依赖的转录终止子。茎环结构具有稳定microRNA的作用，使大多数microRNA稳定性明显大于mRNA。与真核细胞microRNA不同，原核细胞microRNA多与靶mRNA的5′端序列互补配对，且碱基互补配对方式也有所不同，与靶mRNA结合的microRNA随靶mRNA一起降解。

3 真核或原核细胞microRNA作用机制

microRNA属于反式作用因子，其主要作用是抑制靶mRNA翻译或引起靶mRNA降解，在转录后水平下调基因表达，一种microRNA可调控多种mRNA，多种microRNA也可协同调控同一mRNA，甚至还能与TF经前馈环(feed-forward loops，FFLs)协同调节同一基因的表达[20-22]。

3.1 真核细胞microRNA作用机制[21-24]真核细胞microRNA抑制基因表达机制：①抑制mRNA翻译：常见于microRNA与靶mRNA互补配对程度较低者；②引起靶mRNA降解：常见于microRNA与靶mRNA互补配对程度较高者。microRNA与靶mRNA不完全互补时，其结合位点通常位于靶mRNA的3′端非翻译区，可通过影响靶mRNA稳定性、干扰靶mRNA与核糖体结合等方式抑制mRNA翻译。microRNA与靶mRNA完全互补时，其结合位点通常位于靶mRNA编码区(encoding region，ER)或开放阅读框(open reading frame，ORF)中，与靶位点结合后引起靶mRNA的降解。植物microRNA大多与靶mRNA互补配对程度较高，动物microRNA大多与靶mRNA互补配对程度较低。

3.2 原核细胞microRNA作用机制[25-31]原核细胞microRNA可通过与靶mRNA碱基互补配对、与某些蛋白相互作用、RNA伴侣蛋白Hfq连接等方式在转录后水平调控基因表达。

3.2.1 microRNA-mRNA碱基互补配对 原核细胞 microRNA与靶mRNA碱基互补配可抑制或促进靶mRNA翻译。多数细菌microRNA通过碱基不完全互补配对与靶mRNA的5′端核糖体结合位点(ribosome binding site，RBS)结合、导致mRNA的稳定性降低或与5′端第5个密码子结合，从而抑制mRNA翻译。少数细菌microRNA结合于靶mRNA的3′端而使mRNA稳定性增强，或结合于靶mRNA茎环结构使之打开，从而起促进mRNA翻译的作用。

3.2.2 microRNA-蛋白相互作用[32-33]原核细胞microRNA与某些胞内蛋白相互作用后可在转录后水平上调或下调靶基因表达。例如，大肠埃希菌、枯草芽胞杆菌(Bacillussubtilis)和恶臭假单胞菌(Pseudomonasputida)一些microRNA与蛋白结合后模拟核酸底物而抑制靶mRNA翻译。此外还发现，大肠埃希菌RNA结合蛋白CsrA能与靶mRNA中RBS结合，阻断该mRNA与核糖体的结合，从而抑制mRNA翻译；CsrB或CsrC RNA可与CsrA蛋白结合，使CsrA蛋白不能与靶mRNA结合，从而发挥促进靶mRNA翻译的作用。

3.2.3 RNA伴侣蛋白Hfq 多数原核细胞microRNA需RNA伴侣蛋白Hfq来增强其与靶mRNA的亲和力以及对核酸酶的抵抗力。例如，大肠埃希菌Hfq以六聚体形式分别与microRNA及其靶mRNA结合，通过影响microRNA二级结构或改变microRNA与靶mRNA的局部浓度，从而提高microRNA对靶mRNA抑制作用甚至引起靶mRNA降解，该结合过程中microRNA的polyU尾和mRNA的U富集序列对Hfq连接功能至关重要[33-34]。

4 原核细胞microRNA-mRNA相互作用位点

细菌等原核细胞mRNA翻译起始时需一个与mRNA结合的核糖体30S亚基、fMET-tRNAfMet和起始因子组成的起始复合物。位于mRNA起始密码子上游富含嘌呤的SD序列(Shine-Dalgarno sequence)在mRNA捕获核糖体30S亚基中起关键作用，该序列与16S核糖体RNA 3′端反义SD序列结合，促进mRNA翻译[35-39]。细菌microRNA大多为反义RNA，作用于正义靶mRNA。细菌等原核细胞microRNA通常与靶mRNA 5′-UTR结合，抑制靶mRNA翻译[38-39]。

4.1 microRNA-mRNA结合时mRNA的作用位点[38-41]细菌等原核细胞microRNA可与靶mRNA的SD序列碱基互补配对，使SD序列不能与核糖体30S亚基结合。有研究发现，大肠埃希菌和沙门菌microRNA与靶mRNA碱基互补配对位点位于mRNA RBS或其附近，若碱基互补配对位点超出起始密码子上游70 或下游15 nt，microRNA抑制作用明显减弱；此外，靶mRNA SD序列和起始密码子未被结合时microRNA才能发挥作用。

4.2 microRNA-mRNA结合时microRNA的作用位点[41-42]肠道菌群microRNA具有如下模块化特征：①具有富含U的3′端区域：促进不依赖Rho因子的转录终止并保护microRNA免受3′核酸外切酶的降解；②具有与Hfq蛋白结合的区域：通过与Hfq蛋白的结合来提高microRNA稳定性；③具有与靶mRNA结合的区域：该区域序列非常保守，含有与靶mRNA结合的种子序列(seed sequence，SS)。目前发现，细菌microRNA均含有SS且位于microRNA的5′端，不同细菌microRNA的SS长度虽有差异，但通常为6～12个核苷酸。

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Advance in research of characteristics and action mechanism of microRNAs from prokaryotes

ZHANG Xin-wei1,3,HUANG Yan-ying1,3,YAN Jie2,SUN Ai-hua3

(1.SchoolofLaboratoryMedicine,WenzhouMedicalUniversity,Wenzhou325035,China; 2.DepartmentofMedicalMicrobiologyandParasitology,SchoolofMedicine,ZhejiangUniversity,Hangzhou310058,China; 3.FacultyofBasicMedicine,HangzhouMedicalCollege,Hangzhou310053,China)

microRNAs is a group of small non-coding RNAs that play a negative regulation role in expression of target genes at post-transcriptional level by inhibition or degradation of target mRNAs after combination of the seed sequence (SS) in microRNAs with the SS-binding sequences usually located at 5′ ends of target mRNAs. microRNAs was firstly found inCaenorhabditiselegans. Subsequently,many different microRNAs in eukaryocytes were revealed. In eukaryocytes,microRNA precursors are transcribed at first and then become functional microRNAs with 21-23 nt in size after splice. Most of eukaryocytic microRNAs combime with the sequences at 3′ end of target mRNAs that cause the translation inhibition or degradation of the mRNAs. In the recent years,many different prokaryocytes,such as bacteria,have been confirmed to possess microRNAs. However,the microRNAs in prokaryotes such as bacteria are 50-400 nt in size and have the biological activity without splice. Moreover,the characteristics,action sites and mechanisms of the prokaryotic microRNAs have some certain diversity compared to the eukaryotic microRNAs. Our review briefly introduce the major regulation mechanisms of gene expression as well as the general characteristics of microRNAs and their regulation mechanisms of gene expression in prokaryocytes and eukaryocytes,which will provide a basis for further and profound study on the gene expression regulation and pathogenic mechanisms of prokaryotic microbial pathogens.

prokaryotes; gene expression; regulation; microRNA; action mechanism

10.3969/j.issn.1002-2694.2017.05.012

国家自然科学基金(81271893)；浙江省卫生高层次创新人才培养工程项目(2012-241)

孙爱华，sunah123@126.com

1.温州医科大学检验医学院，温州 325035； 2.浙江大学医学院病原生物学系，杭州 310058； 3.杭州医学院基础医学部，杭州 310053

R394.8

A

1002-2694(2017)05-0449-05

2016-11-11 编辑：王晓欢