原核細(xì)胞microRNA特性及其作用機(jī)制研究進(jìn)展

張新蔚，黃燕穎，嚴(yán) 杰，孫愛(ài)華

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原核細(xì)胞microRNA特性及其作用機(jī)制研究進(jìn)展

張新蔚1,3，黃燕穎1,3，嚴(yán) 杰2，孫愛(ài)華3

microRNA為一類(lèi)非編碼小RNA，主要通過(guò)其種子序列(seed sequence，SS)與靶mRNA位于5′端的SS結(jié)合序列特異性結(jié)合后抑制靶mRNA轉(zhuǎn)錄或降解靶mRNA，從而在轉(zhuǎn)錄后水平負(fù)調(diào)控靶基因表達(dá)。microRNA首先發(fā)現(xiàn)于秀麗隱桿線蟲(chóng)(Caenorhabditiselegans)，此后發(fā)現(xiàn)各種真核細(xì)胞均存在大量各種microRNA。真核細(xì)胞首先轉(zhuǎn)錄出microRNA前體，經(jīng)剪切后成為21～23 nt有功能的microRNA，大多與靶mRNA的3′端序列結(jié)合后引起靶mRNA翻譯抑制或降解。近年不少細(xì)菌等原核細(xì)胞microRNA被發(fā)現(xiàn)，但原核細(xì)胞microRNA不需剪切即有活性，大小為50～400 nt，其特性、作用位點(diǎn)和機(jī)制等也與真核細(xì)胞有一定差異。本文簡(jiǎn)要介紹真核和原核細(xì)胞基因表達(dá)調(diào)控主要機(jī)制、microRNA特點(diǎn)及其調(diào)控基因表達(dá)的機(jī)制，以期為深入研究原核細(xì)胞型病原微生物基因表達(dá)調(diào)控與致病機(jī)制奠定基礎(chǔ)。

原核細(xì)胞；基因表達(dá)；調(diào)控；microRNA；作用機(jī)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、核酸及其復(fù)合物，在生命體生理或病理過(guò)程中發(fā)揮關(guān)鍵作用。基因是DNA分子中攜帶遺傳信息的片段，其表達(dá)產(chǎn)物為蛋白或多肽。酶是一類(lèi)具有催化功能的蛋白，上述生物大分子合成與降解均依賴(lài)于酶的作用。因此，了解基因表達(dá)調(diào)控的物質(zhì)基礎(chǔ)及其工作機(jī)制具有重要意義?；虮磉_(dá)過(guò)程分為mRNA轉(zhuǎn)錄和翻譯(轉(zhuǎn)錄后)兩大環(huán)節(jié)。轉(zhuǎn)錄因子(transcription factor，TF)是能與DNA結(jié)合并在轉(zhuǎn)錄水平上靶基因表達(dá)的蛋白，但近年發(fā)現(xiàn)一類(lèi)稱(chēng)之為microRNA的非編碼小RNA也能在轉(zhuǎn)錄后水平上調(diào)控靶基因表達(dá)。本文簡(jiǎn)要介紹真核或原核細(xì)胞TF尤其是microRNA調(diào)控靶基因表達(dá)的主要機(jī)制及其差異。

1 TF調(diào)控靶基因表達(dá)的主要機(jī)制

一般認(rèn)為，真核或原核細(xì)胞基因表達(dá)調(diào)控主要依賴(lài)于TF，microRNA通常僅有微調(diào)作用。一個(gè)基因編碼的蛋白對(duì)該基因或另一基因表達(dá)的調(diào)節(jié)作用分別稱(chēng)為順式或反式調(diào)節(jié)作用，包括基因轉(zhuǎn)錄的開(kāi)啟和關(guān)閉。

1.1 真核細(xì)胞TF調(diào)控靶基因表達(dá)機(jī)制[1-7]在真核細(xì)胞中，順式作用元件(cis-acting element)和反式作用因子(trans-acting factor)及其相互作用是轉(zhuǎn)錄水平調(diào)控基因表達(dá)的基本機(jī)制。順式作用元件為位于基因旁側(cè)、可調(diào)控或影響該基因表達(dá)的特殊序列，根據(jù)功能不同可分為啟動(dòng)子(promotor)、增強(qiáng)子(enhanser)、沉默子(silencer)應(yīng)答元件(response element)。反式作用因子能直接或間接識(shí)別并結(jié)合另一基因順式作用元件核心序列、調(diào)控該基因轉(zhuǎn)錄水平或過(guò)程，以TF最為重要和常見(jiàn)。TF特異性識(shí)別靶基因啟動(dòng)子區(qū)中TF結(jié)合位點(diǎn)(TF-binding site，TFBS)并與之結(jié)合，激活或抑制基因轉(zhuǎn)錄。多個(gè)TFBS可組成順式調(diào)節(jié)模塊(cis-regulatory modules，CRM)，大多數(shù)基因表達(dá)由多種TF通過(guò)CRM調(diào)控，一個(gè)TF也可通過(guò)不同的CRM調(diào)控多個(gè)基因表達(dá)。根據(jù)功能不同，TF可分為轉(zhuǎn)錄激活子(transcription activator)和后者稱(chēng)轉(zhuǎn)錄抑制子(transcription inhibitor)，前者為增強(qiáng)子結(jié)合蛋白(enhancer-binding protein，EBP)，與增強(qiáng)子序列結(jié)合后上調(diào)基因表達(dá)，后者屬于沉默子結(jié)合蛋白(silencer-binding protein，SBP)，與沉默子序列結(jié)合后下調(diào)基因表達(dá)。增強(qiáng)子和沉默子序列位于轉(zhuǎn)錄起始點(diǎn)一定距離外(1-30 kb)，其作用不受序列方向的影響，也可對(duì)異源基因發(fā)揮作用。此外，TF對(duì)某一靶基因表現(xiàn)為表達(dá)上調(diào)作用，但對(duì)另一靶基因可呈現(xiàn)為表達(dá)抑制作用。

1.2 原核細(xì)胞基因表達(dá)調(diào)控主要機(jī)制[6-11]大多數(shù)原核細(xì)胞通過(guò)操縱子調(diào)控基因表達(dá)。操縱子由結(jié)構(gòu)基因和調(diào)控序列組成，前者常為功能上有關(guān)聯(lián)、串聯(lián)排列的數(shù)個(gè)基因，共同構(gòu)成編碼區(qū)，后者包括啟動(dòng)子、操縱元件(operator)以及一定距離外的調(diào)節(jié)基因。操縱元件是一段能被特異的阻遏蛋白(repressor)識(shí)別并結(jié)合的DNA序列，與啟動(dòng)子序列毗鄰甚至重疊。阻遏蛋白與操縱子結(jié)合后，阻斷RNA聚合酶與啟動(dòng)子結(jié)合或阻礙RNA聚合酶沿DNA向前移動(dòng)，在轉(zhuǎn)錄水平上抑制基因表達(dá)。許多原核細(xì)胞基因操縱子中還有一段獨(dú)特的DNA序列，與激活蛋白(activator)結(jié)合后增強(qiáng)RNA聚合酶活性，在轉(zhuǎn)錄水平上調(diào)基因表達(dá)。調(diào)節(jié)基因編碼能與操縱序列結(jié)合的調(diào)控蛋白(modulin)，可分為特異因子、阻遏蛋白和激活蛋白三類(lèi)，其中特異因子決定RNA聚合酶對(duì)一個(gè)或一套啟動(dòng)子序列的特異性識(shí)別和結(jié)合能力。

任何生物體必然與其生存環(huán)境發(fā)生相互聯(lián)系并受之影響，由信號(hào)傳導(dǎo)系統(tǒng)(signaling system)感受外界信號(hào)并作出應(yīng)答，其中跨膜感受器(sensor)蛋白激酶接受環(huán)境分子信號(hào)并向胞內(nèi)傳遞，最終通過(guò)TF調(diào)控相關(guān)基因表達(dá)來(lái)應(yīng)對(duì)環(huán)境變化。與真核細(xì)胞比較，細(xì)菌等原核細(xì)胞信號(hào)傳導(dǎo)系統(tǒng)相對(duì)簡(jiǎn)單，一般由兩類(lèi)分工明確的蛋白組成，故稱(chēng)之二元信號(hào)傳導(dǎo)系統(tǒng)(two-component signaling system，TCSS)：①跨膜傳感器蛋白(sensor protein，SP)：多為組氨酸激酶，少數(shù)為絲氨酸/蘇氨酸激酶；②胞漿內(nèi)應(yīng)答調(diào)節(jié)蛋白(response regulator protein，RRP)，通常被跨膜傳感激酶磷酸化后激活，具有類(lèi)似真核細(xì)胞TF功能，通過(guò)調(diào)節(jié)靶基因表達(dá)水平對(duì)環(huán)境信號(hào)進(jìn)行適應(yīng)性應(yīng)答。例如，金黃色葡萄球菌(Staphylococcusaureus)ArlRS是調(diào)控許多毒力基因表達(dá)的TCSS，ArlS為SP、ArlR為RRP，ArlR上調(diào)sdrC/D/rE、tcaB和ssaA等毒力基因表達(dá)，但下調(diào)lukD/E、phlC和hlgC毒素基因表達(dá)；大腸埃希菌(Escherichiacoli)EnvZ/OmpR 是環(huán)境滲透壓TCSS，EnvZ為SP、OmpR為RRP，OmpR可分別與外膜孔蛋白OmpF和OmpC編碼基因啟動(dòng)子區(qū)結(jié)合，低滲時(shí)OmpF表達(dá)上調(diào)、OmpC表達(dá)下調(diào)，高滲時(shí)OmpF和OmpC表達(dá)水平相反。

2 真核或原核細(xì)胞microRNA產(chǎn)生及特性

microRNA首先發(fā)現(xiàn)于秀麗隱桿線蟲(chóng)(Caenorhabditiselegans)，此后發(fā)現(xiàn)真核或原核細(xì)胞均存在大量各種microRNA[12-13]。真核或原核細(xì)胞microRNA產(chǎn)生過(guò)程、分子大小與結(jié)構(gòu)以及作用機(jī)制均存在一定差異。

2.1 真核細(xì)胞microRNA產(chǎn)生及特性[14-16]在RNA聚合酶的作用下，真核細(xì)胞microRNA編碼基因轉(zhuǎn)錄產(chǎn)生pri-microRNA，隨后Drosha 酶將pri-microRNA剪切成pre-microRNA，然后Ran-GTP 和 Exportin-5蛋白將pre-microRNA從細(xì)胞核運(yùn)送到細(xì)胞質(zhì)，再經(jīng)胞漿Dicer核酸酶剪切成通常為21～23 nt左右成熟的單鏈小RNA(microRNA)。胞漿內(nèi)RNA誘導(dǎo)沉默復(fù)合體(RNA-induce silencing complex，RISC)可識(shí)別microRNA并將其遞呈至靶mRNA，大多數(shù)microRNA與靶mRNA的3’端非翻譯區(qū)(untranslated region，UTR)序列互補(bǔ)，抑制該mRNA翻譯或引起mRNA降解。動(dòng)物約有1/3基因受到microRNA調(diào)控。

2.2 原核細(xì)胞microRNA產(chǎn)生及特性[17-20]原核細(xì)胞microRNA長(zhǎng)度為50～400 nt，轉(zhuǎn)錄后一般不經(jīng)過(guò)加工即有活性。原核細(xì)胞microRNA起始一端折疊成莖環(huán)結(jié)構(gòu)，轉(zhuǎn)錄終止于一個(gè)Rho不依賴(lài)的轉(zhuǎn)錄終止子。莖環(huán)結(jié)構(gòu)具有穩(wěn)定microRNA的作用，使大多數(shù)microRNA穩(wěn)定性明顯大于mRNA。與真核細(xì)胞microRNA不同，原核細(xì)胞microRNA多與靶mRNA的5′端序列互補(bǔ)配對(duì)，且堿基互補(bǔ)配對(duì)方式也有所不同，與靶mRNA結(jié)合的microRNA隨靶mRNA一起降解。

3 真核或原核細(xì)胞microRNA作用機(jī)制

microRNA屬于反式作用因子，其主要作用是抑制靶mRNA翻譯或引起靶mRNA降解，在轉(zhuǎn)錄后水平下調(diào)基因表達(dá)，一種microRNA可調(diào)控多種mRNA，多種microRNA也可協(xié)同調(diào)控同一mRNA，甚至還能與TF經(jīng)前饋環(huán)(feed-forward loops，F(xiàn)FLs)協(xié)同調(diào)節(jié)同一基因的表達(dá)[20-22]。

3.1 真核細(xì)胞microRNA作用機(jī)制[21-24]真核細(xì)胞microRNA抑制基因表達(dá)機(jī)制：①抑制mRNA翻譯：常見(jiàn)于microRNA與靶mRNA互補(bǔ)配對(duì)程度較低者；②引起靶mRNA降解：常見(jiàn)于microRNA與靶mRNA互補(bǔ)配對(duì)程度較高者。microRNA與靶mRNA不完全互補(bǔ)時(shí)，其結(jié)合位點(diǎn)通常位于靶mRNA的3′端非翻譯區(qū)，可通過(guò)影響靶mRNA穩(wěn)定性、干擾靶mRNA與核糖體結(jié)合等方式抑制mRNA翻譯。microRNA與靶mRNA完全互補(bǔ)時(shí)，其結(jié)合位點(diǎn)通常位于靶mRNA編碼區(qū)(encoding region，ER)或開(kāi)放閱讀框(open reading frame，ORF)中，與靶位點(diǎn)結(jié)合后引起靶mRNA的降解。植物microRNA大多與靶mRNA互補(bǔ)配對(duì)程度較高，動(dòng)物microRNA大多與靶mRNA互補(bǔ)配對(duì)程度較低。

3.2 原核細(xì)胞microRNA作用機(jī)制[25-31]原核細(xì)胞microRNA可通過(guò)與靶mRNA堿基互補(bǔ)配對(duì)、與某些蛋白相互作用、RNA伴侶蛋白Hfq連接等方式在轉(zhuǎn)錄后水平調(diào)控基因表達(dá)。

3.2.1 microRNA-mRNA堿基互補(bǔ)配對(duì) 原核細(xì)胞 microRNA與靶mRNA堿基互補(bǔ)配可抑制或促進(jìn)靶mRNA翻譯。多數(shù)細(xì)菌microRNA通過(guò)堿基不完全互補(bǔ)配對(duì)與靶mRNA的5′端核糖體結(jié)合位點(diǎn)(ribosome binding site，RBS)結(jié)合、導(dǎo)致mRNA的穩(wěn)定性降低或與5′端第5個(gè)密碼子結(jié)合，從而抑制mRNA翻譯。少數(shù)細(xì)菌microRNA結(jié)合于靶mRNA的3′端而使mRNA穩(wěn)定性增強(qiáng)，或結(jié)合于靶mRNA莖環(huán)結(jié)構(gòu)使之打開(kāi)，從而起促進(jìn)mRNA翻譯的作用。

3.2.2 microRNA-蛋白相互作用[32-33]原核細(xì)胞microRNA與某些胞內(nèi)蛋白相互作用后可在轉(zhuǎn)錄后水平上調(diào)或下調(diào)靶基因表達(dá)。例如，大腸埃希菌、枯草芽胞桿菌(Bacillussubtilis)和惡臭假單胞菌(Pseudomonasputida)一些microRNA與蛋白結(jié)合后模擬核酸底物而抑制靶mRNA翻譯。此外還發(fā)現(xiàn)，大腸埃希菌RNA結(jié)合蛋白CsrA能與靶mRNA中RBS結(jié)合，阻斷該mRNA與核糖體的結(jié)合，從而抑制mRNA翻譯；CsrB或CsrC RNA可與CsrA蛋白結(jié)合，使CsrA蛋白不能與靶mRNA結(jié)合，從而發(fā)揮促進(jìn)靶mRNA翻譯的作用。

3.2.3 RNA伴侶蛋白Hfq 多數(shù)原核細(xì)胞microRNA需RNA伴侶蛋白Hfq來(lái)增強(qiáng)其與靶mRNA的親和力以及對(duì)核酸酶的抵抗力。例如，大腸埃希菌Hfq以六聚體形式分別與microRNA及其靶mRNA結(jié)合，通過(guò)影響microRNA二級(jí)結(jié)構(gòu)或改變microRNA與靶mRNA的局部濃度，從而提高microRNA對(duì)靶mRNA抑制作用甚至引起靶mRNA降解，該結(jié)合過(guò)程中microRNA的polyU尾和mRNA的U富集序列對(duì)Hfq連接功能至關(guān)重要[33-34]。

4 原核細(xì)胞microRNA-mRNA相互作用位點(diǎn)

細(xì)菌等原核細(xì)胞mRNA翻譯起始時(shí)需一個(gè)與mRNA結(jié)合的核糖體30S亞基、fMET-tRNAfMet和起始因子組成的起始復(fù)合物。位于mRNA起始密碼子上游富含嘌呤的SD序列(Shine-Dalgarno sequence)在mRNA捕獲核糖體30S亞基中起關(guān)鍵作用，該序列與16S核糖體RNA 3′端反義SD序列結(jié)合，促進(jìn)mRNA翻譯[35-39]。細(xì)菌microRNA大多為反義RNA，作用于正義靶mRNA。細(xì)菌等原核細(xì)胞microRNA通常與靶mRNA 5′-UTR結(jié)合，抑制靶mRNA翻譯[38-39]。

4.1 microRNA-mRNA結(jié)合時(shí)mRNA的作用位點(diǎn)[38-41]細(xì)菌等原核細(xì)胞microRNA可與靶mRNA的SD序列堿基互補(bǔ)配對(duì)，使SD序列不能與核糖體30S亞基結(jié)合。有研究發(fā)現(xiàn)，大腸埃希菌和沙門(mén)菌microRNA與靶mRNA堿基互補(bǔ)配對(duì)位點(diǎn)位于mRNA RBS或其附近，若堿基互補(bǔ)配對(duì)位點(diǎn)超出起始密碼子上游70 或下游15 nt，microRNA抑制作用明顯減弱；此外，靶mRNA SD序列和起始密碼子未被結(jié)合時(shí)microRNA才能發(fā)揮作用。

4.2 microRNA-mRNA結(jié)合時(shí)microRNA的作用位點(diǎn)[41-42]腸道菌群microRNA具有如下模塊化特征：①具有富含U的3′端區(qū)域：促進(jìn)不依賴(lài)Rho因子的轉(zhuǎn)錄終止并保護(hù)microRNA免受3′核酸外切酶的降解；②具有與Hfq蛋白結(jié)合的區(qū)域：通過(guò)與Hfq蛋白的結(jié)合來(lái)提高microRNA穩(wěn)定性；③具有與靶mRNA結(jié)合的區(qū)域：該區(qū)域序列非常保守，含有與靶mRNA結(jié)合的種子序列(seed sequence，SS)。目前發(fā)現(xiàn)，細(xì)菌microRNA均含有SS且位于microRNA的5′端，不同細(xì)菌microRNA的SS長(zhǎng)度雖有差異，但通常為6～12個(gè)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

國(guó)家自然科學(xué)基金(81271893)；浙江省衛(wèi)生高層次創(chuàng)新人才培養(yǎng)工程項(xiàng)目(2012-241)

孫愛(ài)華，sunah123@126.com

1.溫州醫(yī)科大學(xué)檢驗(yàn)醫(yī)學(xué)院，溫州 325035； 2.浙江大學(xué)醫(yī)學(xué)院病原生物學(xué)系，杭州 310058； 3.杭州醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)部，杭州 310053

R394.8

A

1002-2694(2017)05-0449-05

2016-11-11 編輯：王曉歡