馬劍峰，甘麥鄰，朱礪，沈林園

轉(zhuǎn)運(yùn)RNA衍生的小RNA功能及其研究方法

馬劍峰1,2，甘麥鄰1,2，朱礪1,2，沈林園1,2

1. 四川農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，成都 611130 2. 四川農(nóng)業(yè)大學(xué)，畜禽遺傳資源發(fā)掘與創(chuàng)新利用四川省重點(diǎn)實(shí)驗(yàn)室，成都 611130

轉(zhuǎn)運(yùn)RNA衍生的小RNA (tRNA-derived small RNAs, tsRNA)是近年來發(fā)現(xiàn)的一類由成熟tRNA或tRNA前體通過特殊的作用機(jī)制加工產(chǎn)生的非編碼RNA。根據(jù)其產(chǎn)生方式的不同，主要分為tRNA半分子(tRNA halves)和tRNA衍生片段(tRNA-derived RNA fragments, tRFs)兩種類型。tsRNA廣泛存在于各種生物體中，具有高度保守、結(jié)構(gòu)穩(wěn)定和組織表達(dá)特異性等特點(diǎn)。大量研究表明，多種細(xì)胞應(yīng)激反應(yīng)中tsRNA的表達(dá)顯著上升，其參與應(yīng)激反應(yīng)的調(diào)控是一種保守的生物現(xiàn)象。tsRNA可通過調(diào)控轉(zhuǎn)錄本穩(wěn)定性、調(diào)節(jié)翻譯和作為表觀遺傳調(diào)控因子等方式在多種生物學(xué)過程中發(fā)揮重要作用，此外，tsRNA具有作為疾病生物標(biāo)志物和治療靶點(diǎn)的潛力，逐漸成為生物醫(yī)學(xué)領(lǐng)域中的研究熱點(diǎn)。本文從tsRNA的生物發(fā)生、功能和研究方法3個(gè)方面，對(duì)其研究現(xiàn)狀進(jìn)行了綜述，以期為后續(xù)研究提供參考。

非編碼RNA；轉(zhuǎn)運(yùn)RNA衍生片段；tsRNA；tRFs

人類基因組中編碼蛋白質(zhì)的基因僅占基因組序列的2%左右，而絕大多數(shù)轉(zhuǎn)錄本不編碼蛋白質(zhì)，研究表明至少有80%的序列被轉(zhuǎn)錄為非編碼RNA (non-coding RNA, ncRNA)[1]。ncRNA包括核糖體RNA (ribosomal RNA, rRNA)、轉(zhuǎn)運(yùn)RNA (transfer RNA, tRNA)、microRNA、小干擾RNA (small interfering RNA, siRNA)、長(zhǎng)鏈非編碼RNA (long non-coding RNA, lncRNA)等[2]，自20世紀(jì)50年代rRNA和tRNA被發(fā)現(xiàn)以來，ncRNA的功能開始被重視。rRNA和tRNA是中心法則的主要參與者，在蛋白質(zhì)合成中發(fā)揮識(shí)別和轉(zhuǎn)運(yùn)的重要作用。隨著高通量測(cè)序技術(shù)的快速發(fā)展，在動(dòng)植物、細(xì)菌和真菌中都鑒定出一種長(zhǎng)度大約為15～50 nt的ncRNA，因?yàn)閬碓从趖RNA，所以將其稱為tRNA-derived small RNAs (tsRNA)；tsRNA結(jié)構(gòu)穩(wěn)定，保守性強(qiáng)，具有豐富的RNA修飾，在各種生物體中廣泛表達(dá)[3～5]。

1977年，tsRNA最初由Borek等[6]在癌癥患者的尿液中檢測(cè)到，并且研究者們將tRNA分解產(chǎn)生的帶有修飾的核苷酸作為癌癥標(biāo)志物[7]，但當(dāng)時(shí)tsRNA被認(rèn)為是tRNA隨機(jī)降解的產(chǎn)物。1992年，Saxena等[8]將血管生成素(Angiogenin, ANG)注射到非洲爪蛙()卵母細(xì)胞中，發(fā)現(xiàn)細(xì)胞中tRNA被降解，而rRNA和mRNA并沒有被降解，證明了ANG是一種特異性水解tRNA的核酸酶。Thompson等[9]在氧化應(yīng)激的酵母菌()、擬南芥()和HeLa細(xì)胞中檢測(cè)到tsRNA的表達(dá)豐度增加，表明tRNA裂解是真核細(xì)胞應(yīng)對(duì)氧化應(yīng)激的一種保守的現(xiàn)象。直到2009年，tsRNA的產(chǎn)生機(jī)制才被初步揭示，F(xiàn)u等[10]在哺乳動(dòng)物細(xì)胞系(HepG2、HeLa、HEK293等)中發(fā)現(xiàn)，多種應(yīng)激條件可以誘導(dǎo)血管生成素裂解tRNA的反密碼子環(huán)產(chǎn)生tsRNA。

隨著tsRNA產(chǎn)生機(jī)制的發(fā)現(xiàn)，tsRNA逐漸被證明參與多種調(diào)控過程，例如調(diào)節(jié)mRNA穩(wěn)定性、翻譯、rRNA合成、RNA逆轉(zhuǎn)錄等，進(jìn)而調(diào)節(jié)細(xì)胞增殖、凋亡、遷移、周期等，在動(dòng)物正常發(fā)育和疾病發(fā)生過程中發(fā)揮生物學(xué)功能[3]。tsRNA的表達(dá)具有組織和時(shí)空特異性，其異常表達(dá)可作為判斷是否發(fā)生疾病的標(biāo)志。但是，目前關(guān)于tsRNA的研究還處于起步階段，其產(chǎn)生機(jī)制和具體生物學(xué)功能還沒有被充分解析，其在正常發(fā)育和疾病發(fā)生過程中的作用還有待進(jìn)一步的深入挖掘。本文總結(jié)了tsRNA的形成機(jī)制、生物學(xué)功能以及相關(guān)研究方法，以期為后續(xù)相關(guān)研究提供參考和借鑒。

1 tsRNA來源及命名原則

在早期的研究中，tsRNA被定義為RNA噪音，因此在測(cè)序數(shù)據(jù)的加工處理過程中被直接剔除掉。然而，隨著對(duì)ncRNA的進(jìn)一步理解和認(rèn)識(shí)，同時(shí)生物信息學(xué)分析手段的不斷提高，研究者們發(fā)現(xiàn)這些曾經(jīng)被認(rèn)為是垃圾序列的tsRNA片段其實(shí)是tRNA的降解產(chǎn)物，并不隨機(jī)產(chǎn)生，可能發(fā)揮著特定的生物學(xué)功能。成熟tRNA以及前體tRNA在相關(guān)核酸酶的作用下通過斷裂的方式形成短鏈的tsRNA。在真核生物中，tRNA基因由RNA聚合酶III轉(zhuǎn)錄生成tRNA前體(pre-tRNA)[11,12]，成熟過程中tRNA前體的5?前導(dǎo)序列和3?尾巴序列分別被核糖核酸酶P (RNase P)和核糖核酸酶Z (RNase Z)切割，隨后在tRNA核苷酸轉(zhuǎn)移酶的作用下，在3?端加上“CCA”序列[13]，經(jīng)過進(jìn)一步堿基修飾和折疊等加工過程形成成熟的tRNA。當(dāng)細(xì)胞在饑餓、缺氧、高溫、氧化損傷等應(yīng)激條件下[14]，特定的核酸酶能對(duì)tRNA特定位點(diǎn)進(jìn)行剪切形成具有功能性的tsRNA分子(表1)。對(duì)于tsRNA的命名目前還沒有規(guī)范的標(biāo)準(zhǔn)，主要根據(jù)其生物學(xué)來源以及斷裂位點(diǎn)命名，通?？梢苑譃閮煞N類型：tRNA半分子(tRNA-halves)和tRNA衍生片段(tRNA-derived fragments, tRFs)[14,24～26]。

表1 應(yīng)激誘導(dǎo)的相關(guān)tsRNA形成

在哺乳動(dòng)物中tRNA-halves是在應(yīng)激反應(yīng)下由血管生成素特異性地剪切成熟體tRNA反密碼子環(huán)形成的28～36 nt的小RNA[10,27]，這一過程受到tRNA修飾的調(diào)控因此只能切割特定位點(diǎn)產(chǎn)生tRNA- halves[28]，它也被稱為tRNA-drived stress-induced small RNA (tiRNA)，根據(jù)其對(duì)應(yīng)的成熟tRNA序列可以分為5?-tiRNA和3?-tiRNA。此外，在氧化應(yīng)激過程中，酵母中的Rny1p酶能促進(jìn)tRNA的裂解[15]，大腸桿菌中PrrC、大腸桿菌素D和E5也能特異性的切割tRNA的反密碼子環(huán)[29]。

tRFs的長(zhǎng)度為15～32 nt，根據(jù)其斷裂位點(diǎn)可以分為5種類型[30,31]。如圖1所示，tRF-1是前體tRNA在成熟過程中由RNase Z或ELAC2剪切3?端形成的片段，因其含poly-U序列又稱為3?U-tRF。tRF-5來自成熟tRNA的5?端，其切割位點(diǎn)在成熟tRNA的D環(huán)或D環(huán)與反密碼子環(huán)之間，可以進(jìn)一步分為3種亞型（tRF-5a、tRF-5b、tRF-5c）。tRF-3由成熟tRNA裂解TΨC環(huán)所產(chǎn)生，其3?末端帶有成熟tRNA的CCA序列，根據(jù)長(zhǎng)度不同可分為tRF-3a和tRF-3b。tRF-2則包含反密碼子環(huán)及其兩端的莖序列，i-tRF (internal tRNA fragments)對(duì)應(yīng)的是成熟tRNA內(nèi)部序列的片段，目前對(duì)這兩種tRFs的研究還很少，其確切的產(chǎn)生機(jī)制還不完全清楚[32～34]。

2 tsRNA生物學(xué)功能

近年來研究表明，tsRNA廣泛存在于細(xì)菌、真菌、動(dòng)植物多物種中，并且在進(jìn)化上具有保守性[35]。tsRNA是具有調(diào)節(jié)功能的小非編碼RNA，通過多種機(jī)制發(fā)揮生物學(xué)作用，包括調(diào)節(jié)mRNA穩(wěn)定性、調(diào)控翻譯過程和作為表觀遺傳調(diào)控因子等。目前，已在不同類型的細(xì)胞系、組織或體液中發(fā)現(xiàn)了大量的tsRNA，相關(guān)研究進(jìn)一步揭示了tsRNA可能參與疾病發(fā)生過程(表2)。

2.1 調(diào)節(jié)mRNA穩(wěn)定性

tsRNA在結(jié)構(gòu)和大小上與microRNA相似。研究早期，部分tsRNA被認(rèn)為是microRNA[62]，miR-1280通過抑制靶基因ROCK1表達(dá)來抑制直腸癌的轉(zhuǎn)移，但后來發(fā)現(xiàn)miR-1208來源于tRNALeu[49]。tsRNA具有microRNA類似的作用，通過靶向mRNA調(diào)節(jié)其穩(wěn)定性。Zhang等[39]在胃癌細(xì)胞中證實(shí)tRF-3019a來源于tRNAAlaAGC，通過靶向FBXO47 mRNA的3?UTR區(qū)降低其表達(dá)。Li等[63]在基因敲除的小鼠胚胎成纖維細(xì)胞中鑒定出tRNAHisGTG和tRNALeuCAG的片段，這些tsRNA不依賴于產(chǎn)生，隨后的研究表明它們可以引導(dǎo)AGO2蛋白裂解靶mRNA。有研究報(bào)道在HEK293細(xì)胞中，5?- tsRNA和3?-tsRNA優(yōu)先與AGO1、AGO3和AGO4結(jié)合，而不與AGO2結(jié)合[64]。Goodarzi等[20]發(fā)現(xiàn)了來自tRNAGlu、tRNAAsp、tRNAGly和tRNATyr的tsRNA，它們通過與RNA結(jié)合蛋白YBX1競(jìng)爭(zhēng)性結(jié)合來抑制乳腺癌細(xì)胞中多種原癌基因轉(zhuǎn)錄本的穩(wěn)定性。5?- tRFGln能夠抑制翻譯，并且不需要與mRNA互補(bǔ)的靶序列，其作用機(jī)制依賴于保守殘基“GG”[65]。因此tsRNA能發(fā)揮microRNA類似的調(diào)控功能，但其具體調(diào)控機(jī)制與microRNA并不完全相同。

圖1 tsRNA分類與作用機(jī)制

2.2 調(diào)控蛋白質(zhì)的翻譯

tsRNA能夠通過多種機(jī)制調(diào)控蛋白質(zhì)的翻譯過程。除了上述調(diào)節(jié)mRNA穩(wěn)定性外，tsRNA還能通過影響核糖體的生物發(fā)生、結(jié)合核糖體、影響翻譯起始過程等調(diào)控翻譯過程。

2.2.1 影響核糖體的生物發(fā)生

核糖體是一個(gè)以rRNA為功能核心的復(fù)合物，核糖體生物發(fā)生是一個(gè)復(fù)雜而精確調(diào)控的細(xì)胞過程，不同的非編碼RNA能參與調(diào)控其rRNA生成[66]。Couvillion等[67]研究表明，tsRNA作為原生動(dòng)物四膜蟲() rRNA剪接復(fù)合體的組成部分參與rRNA合成過程，剪接復(fù)合體中包含3?-tsRNA、Twi12蛋白、Tan1蛋白和外切酶Xrn2，結(jié)合后Twi12穩(wěn)定并激活Xrn2的外切酶活性，進(jìn)而切割前體rRNA。Kim等[68]研究表明，HeLa細(xì)胞中3?-tsRNALeuCAG可以結(jié)合核糖體蛋白R(shí)PS28的mRNA增強(qiáng)其翻譯，RPS28翻譯的減少會(huì)阻斷18S核糖體RNA成熟并誘導(dǎo)細(xì)胞凋亡。隨后在小鼠和人類的研究中也進(jìn)一步證實(shí)了tsRNALeuCAG通過與編碼區(qū)靶位點(diǎn)相互作用調(diào)節(jié)RPS28的翻譯，表明其功能在不同物種中具有保守性[69]。

表2 與疾病相關(guān)的tsRNA

2.2.2 tsRNA與核糖體結(jié)合

tsRNA能與核糖體成分結(jié)合調(diào)控翻譯過程。Gebetsberger等[22]對(duì)古細(xì)菌中與核糖體共同純化的小RNA進(jìn)行測(cè)序，發(fā)現(xiàn)在高pH值條件下生長(zhǎng)的細(xì)胞中5?-tRFVal表達(dá)豐度高，該片段與小核糖體亞基結(jié)合抑制整體翻譯水平。最近的研究發(fā)現(xiàn)[70]，Val-tRF與小核糖體亞基結(jié)合干擾翻譯起始復(fù)合物的形成，將其導(dǎo)入酵母菌和大腸桿菌中也能夠抑制蛋白質(zhì)的合成。布魯氏錐蟲()中的3?-tiRNAThr在古細(xì)菌和酵母菌中通過類似的作用來介導(dǎo)刺激翻譯[19]，這些研究表明，tsRNA與核糖體結(jié)合可能是一種跨物種的保守的調(diào)控機(jī)制。

2.2.3 調(diào)控翻譯起始過程

翻譯調(diào)控主要發(fā)生在翻譯起始水平，因?yàn)檫@是蛋白質(zhì)生物合成中最關(guān)鍵和限速的步驟。細(xì)胞受到應(yīng)激后，真核起始因子eIF2α磷酸化導(dǎo)致應(yīng)激顆粒的形成，可抑制整體蛋白質(zhì)的合成[71]，Yamasaki等[23]研究表明，亞砷酸鹽、熱休克或紫外線照射促進(jìn)了血管生成素切割tRNA，細(xì)胞中tiRNA積累導(dǎo)致不依賴于eIF2α磷酸化的應(yīng)激顆粒形成。Anderson研究小組[72,73]發(fā)現(xiàn)5?-tiRNAAla和5?-tiRNACys在其5?末端帶有4～5個(gè)鳥嘌呤殘基的序列，能夠形成分子間RNA G四聚體(RG4)來代替翻譯起始因子eIF4復(fù)合物，進(jìn)一步研究發(fā)現(xiàn)RG4與eIF4G結(jié)合，破壞40S核糖體對(duì)mRNA的識(shí)別，激活應(yīng)激反應(yīng)中的細(xì)胞保護(hù)作用[71]。此外，YB-1是細(xì)胞質(zhì)中應(yīng)激顆粒的組成部分，RG4結(jié)構(gòu)能與YB-1結(jié)合，抑制翻譯起始復(fù)合物形成[74]。

2.3 作為表觀遺傳調(diào)控因子

表觀遺傳學(xué)指在DNA序列不改變的情況下，基因功能發(fā)生可遺傳變化。表觀遺傳學(xué)主要包括DNA甲基化、乙酰化、組蛋白修飾和ncRNA等，這些表觀遺傳信息和DNA序列信息共同參與調(diào)控基因的表達(dá)[75,76]。大量研究表明，tsRNA可以影響不同的表觀遺傳過程來調(diào)節(jié)基因的表達(dá)。Argonaute蛋白分為AGO和PIWI兩個(gè)家族，其中AGO蛋白廣泛表達(dá)而PIWI主要在生殖細(xì)胞中表達(dá)，一類長(zhǎng)度為24～32 nt的piRNA能與PIWI蛋白形成復(fù)合物，通過表觀遺傳參與基因的調(diào)控[77,78]。tsRNA和piRNA的長(zhǎng)度相似，也可以和PIWI蛋白相互作用來參與表觀遺傳的調(diào)控。Couvillion等[79]證實(shí)了tsRNA與四膜蟲PIWI蛋白Twi12相關(guān)，兩者表達(dá)豐富呈高度正相關(guān)，在之后研究中發(fā)現(xiàn)Twi12-tsRNA復(fù)合物能夠調(diào)控核內(nèi)RNA的加工[67]。Zhang等[80]發(fā)現(xiàn)，tsRNAs可以調(diào)節(jié)人單核細(xì)胞的組蛋白甲基化，這些單核細(xì)胞在IL-4刺激下分化為樹突狀細(xì)胞，在分化的細(xì)胞中鑒定出表達(dá)下調(diào)的piRNA，并與tRNAGlu的5'端序列相匹配，5?-tsRNAGlu與PIWIL4相互作用促進(jìn)H3K9甲基化從而抑制的轉(zhuǎn)錄。動(dòng)植物基因組中含有大量的轉(zhuǎn)座因子，它是一種對(duì)基因組有潛在危害的可移動(dòng)基因組DNA，其轉(zhuǎn)錄活性通常被表觀遺傳標(biāo)記抑制，如DNA甲基化和組蛋白修飾[81]。在著床前的胚胎發(fā)育過程中，大多數(shù)表觀遺傳標(biāo)記消失，此過程中需要其他的機(jī)制來保護(hù)基因組[82]。tsRNA可以通過靶向轉(zhuǎn)座子引物結(jié)合位點(diǎn)，抑制轉(zhuǎn)座子活性。Schorn等[83]發(fā)現(xiàn)3?-tsRNACCA在(SET domain bifurcated histone lysine methyltrans-ferase 1)敲除的胚胎干細(xì)胞中高表達(dá)，而在(DNA methyltransferase 1)敲除的細(xì)胞中沒有。Dnmt1是哺乳動(dòng)物細(xì)胞中主要DNA甲基轉(zhuǎn)移酶，介導(dǎo)DNA甲基化導(dǎo)致非LTR逆轉(zhuǎn)錄轉(zhuǎn)座子的抑制[84]，而Setdb1介導(dǎo)H3K9三甲基化(H3K9me3)在內(nèi)源性和外源性逆轉(zhuǎn)錄轉(zhuǎn)座子沉默中起著關(guān)鍵作用[85]。Schorn等[83]研究表明tsRNA可能作為新的表觀遺傳調(diào)控因子，調(diào)節(jié)LTR逆轉(zhuǎn)錄轉(zhuǎn)座子的表達(dá)。

3 tsRNA研究方法

隨著在更多物種中tsRNA被發(fā)現(xiàn)，深入研究其生物學(xué)功能以及參與的調(diào)控機(jī)制成為新的研究熱點(diǎn)。目前，tsRNA的研究思路主要是通過高通量測(cè)序篩選出組間差異表達(dá)的tsRNA，然后驗(yàn)證確定目標(biāo)分子，圍繞目標(biāo)分子進(jìn)行體內(nèi)外的功能驗(yàn)證和調(diào)控網(wǎng)絡(luò)的構(gòu)建。

3.1 tsRNA相關(guān)數(shù)據(jù)庫(kù)

目前國(guó)內(nèi)外學(xué)者對(duì)tsRNA的研究主要集中在癌癥等疾病方面，tsRNA常作為與microRNA相似的小RNA進(jìn)行研究，從測(cè)序數(shù)據(jù)中鑒別出哪些小RNA屬于tsRNA尤為重要，為此研究者們建立了多個(gè)用于tsRNA研究的數(shù)據(jù)庫(kù)(表3)。Kumar等[86]建立了第一個(gè)tsRNA的數(shù)據(jù)庫(kù)tRFdb，包括了人和小鼠等8個(gè)物種的tsRNA序列，通過序列或ID可以檢索到其相應(yīng)的類型、tRNA基因位置和有關(guān)實(shí)驗(yàn)。tRF2Cancer[87]是一個(gè)用于癌癥研究的數(shù)據(jù)庫(kù)，支持用戶上傳測(cè)序數(shù)據(jù)和CLIP-Seq數(shù)據(jù)，以鑒定tsRNA、預(yù)測(cè)靶標(biāo)和分析生物學(xué)功能等。Schuster等[88]開發(fā)了專門用于多物種精子RNA分析的數(shù)據(jù)庫(kù)SpermBase，可用于精子tsRNA分析。PtRFdb是由Gupta等[89]開發(fā)的植物tsRNA數(shù)據(jù)庫(kù)。Li等[30]開發(fā)的tRFtarget能有效地預(yù)測(cè)tsRNA靶基因的結(jié)合位點(diǎn)、結(jié)合區(qū)域以及穩(wěn)定性，應(yīng)用于tsRNA的功能研究。tsRBase[90]是目前最全面、最系統(tǒng)的tsRNA數(shù)據(jù)庫(kù)，集成了多個(gè)物種的tsRNA表達(dá)模式，該數(shù)據(jù)庫(kù)收集了來自不同組織/細(xì)胞系，或不同處理和遺傳背景下的樣本，有助于描述不同條件下tsRNA的特定表達(dá)模式。這些數(shù)據(jù)庫(kù)能幫助研究者們對(duì)tsRNA進(jìn)行深入研究，但目前仍缺乏對(duì)這些數(shù)據(jù)庫(kù)的準(zhǔn)確性和靈敏性的綜合評(píng)估。

3.2 tsRNA鑒別和定量方法

微陣列和RNA-seq是高通量檢測(cè)tsRNA表達(dá)情況的有效工具[33]。Farina等[36]利用微陣列分析了乳腺上皮細(xì)胞系中的tsRNA表達(dá)變化，檢測(cè)到對(duì)腫瘤抑制因子RUNX1敏感的tsRNA。Wang等[91]對(duì)乳腺癌組織中tsRNA表達(dá)譜進(jìn)行了研究，鑒定了其中30個(gè)顯著差異的tsRNA。Xu等[92]對(duì)多發(fā)性骨髓瘤進(jìn)行了RNA-seq，鑒定出33個(gè)上調(diào)和22個(gè)下調(diào)的tsRNA并對(duì)其功能進(jìn)行了分析，發(fā)現(xiàn)tsRNA可能參與骨髓瘤的發(fā)生。目前使用廣泛的小RNA測(cè)序建庫(kù)流程存在一定缺陷，會(huì)導(dǎo)致測(cè)序結(jié)果產(chǎn)生系統(tǒng)性偏差[93]。與microRNA相比，tsRNA帶有大量的RNA修飾會(huì)妨礙cDNA建庫(kù)過程中5?和3?接頭的連接和逆轉(zhuǎn)錄酶的作用[94]，導(dǎo)致含有特殊修飾的tsRNA無法被檢測(cè)，因此需要將總RNA進(jìn)行預(yù)處理以提高測(cè)序文庫(kù)構(gòu)建的效率。最近，Shi等[95]開發(fā)了PANDORA-seq來克服這一問題。PANDORA-seq主要利用兩種酶(AlkB和T4PNK)處理，將RNA末端的3?-P和2?3?-CP轉(zhuǎn)換為3?-OH，5?-OH轉(zhuǎn)換為5?-P，去掉了部分RNA甲基化，如m1A、m3C、m1G、m22G。其測(cè)序結(jié)果揭示了小鼠組織中與之前研究完全不同的小RNA表達(dá)圖譜，并且能夠通過Northern blot實(shí)驗(yàn)驗(yàn)證。

實(shí)時(shí)熒光定量逆轉(zhuǎn)錄–聚合酶鏈?zhǔn)椒磻?yīng)(real time quantitative reverse transcription-polymerase chain reaction, qRT-PCR)和Northern 印跡(Northern blot)是用來定量tsRNA的最成熟的技術(shù)，常用于驗(yàn)證RNA-seq的結(jié)果。Wang等[96]利用qRT-PCR驗(yàn)證了差異表達(dá)tsRNA在早期乳腺癌患者血漿、組織中的表達(dá)豐度，確定了6個(gè)tsRNA可以作為早期乳腺癌的潛在診斷標(biāo)志物。Shi等[97]去除了總RNA的修飾后，再用qRT-PCR檢測(cè)腎小球足細(xì)胞中部分tsRNA的表達(dá)。此外，Lee等[98]將總RNA進(jìn)行凝膠純化后，把其中17～26 nt的小RNA用于合成cDNA再進(jìn)行PCR，其實(shí)驗(yàn)結(jié)果與總RNA進(jìn)行qRT-PCR結(jié)果一致。與qRT-PCR相比，Northern blot檢測(cè)靈敏度較低，Su等[99]通過Northern blot檢測(cè)到了胎盤中表達(dá)豐度較高tsRNA，驗(yàn)證了RNA-seq結(jié)果的真實(shí)性。McArdle等[100]開發(fā)了一種基于電催化鉑納米顆粒的電化學(xué)直接檢測(cè)方法，能夠特異性的檢測(cè)血漿中3種tsRNA，并且與qPCR獲得的結(jié)果相同，為快速檢測(cè)tsRNA提供了新的研究思路。

3.3 tsRNA過表達(dá)和干擾技術(shù)

過表達(dá)或RNA干擾技術(shù)常被用來進(jìn)行功能獲得性或缺失性研究。通過導(dǎo)入人工合成的帶有修飾的核苷酸來過表達(dá)或抑制目標(biāo)tsRNA，觀察細(xì)胞、動(dòng)物表型的變化或靶基因、靶蛋白的變化，是目前研究tsRNA功能的常用方法。與microRNA研究方法類似，tsRNA mimics和inhibitors是最常用的技術(shù)手段[101]：mimics是運(yùn)用化學(xué)方法合成的雙鏈RNA，能模擬內(nèi)源性tsRNA進(jìn)而增強(qiáng)內(nèi)源性tsRNA的功能，其進(jìn)入細(xì)胞后與Argonaute蛋白結(jié)合形成沉默復(fù)合物，進(jìn)一步與靶基因mRNA結(jié)合調(diào)控其表達(dá)；inhibitor是化學(xué)合成的單鏈RNA，是專門針對(duì)細(xì)胞中特異的靶tsRNA的抑制劑，轉(zhuǎn)染至細(xì)胞后與tsRNA互補(bǔ)結(jié)合，削弱內(nèi)源性tsRNA的效應(yīng)。Shen等[102]對(duì)小鼠3T3-L1前脂肪細(xì)胞瞬時(shí)轉(zhuǎn)染tRFGluTTCmimics或inhibitors，發(fā)現(xiàn)tRFGluTTC具有促進(jìn)脂肪細(xì)胞增殖的功能。鎖核酸(locked-nucleic acids, LNAs)是一種具有雙環(huán)結(jié)構(gòu)的寡核酸衍生物，其2?-O位和4?-C位通過縮水作用形成氧亞甲基橋，由于結(jié)構(gòu)的特殊性其具有與天然核酸的高親和力、堿基錯(cuò)配識(shí)別能力和抗核酸酶解特點(diǎn)[103]。使用LNAs合成的tsRNA抑制劑，在tsRNA的功能研究中取得不錯(cuò)的效果。Goodarzi等[20]使用反義LNAs抑制腫瘤細(xì)胞中的tRFGlu、tRFAsp和tRFGly，導(dǎo)致RNA結(jié)合蛋白YBX1與原癌基因mRNA的結(jié)合更加穩(wěn)定，揭示了tsRNA的腫瘤抑制作用。Kim等[68]用LNA/ASO混合物抑制3?-tsRNALeuCAG，顯著降低了HCT-116細(xì)胞活性，并且證明了LNA并不影響成熟tRNA的功能。

有研究報(bào)道，一些tRNA基因的差異表達(dá)導(dǎo)致tRNA衍生片段豐度的變化，而不是成熟tRNA豐度的變化[104]。此外，通過過表達(dá)親本tRNA實(shí)現(xiàn)了上調(diào)tsRNA的表達(dá)。例如，Green等[105]用tRNA過表達(dá)質(zhì)粒轉(zhuǎn)染軟骨細(xì)胞，使tRF-3003a的水平顯著升高，并且其抑制靶基因的效果和轉(zhuǎn)染mimics相似。Kuscu等[106]的研究中也實(shí)現(xiàn)了過表達(dá)親本tRNA使tRF-3水平升高。與化學(xué)合成的tsRNA類似物相比，親本tRNA產(chǎn)生的內(nèi)源性tsRNA更有利于功能性研究，但過表達(dá)一種tRNA可能會(huì)產(chǎn)生不同類型的tsRNA，對(duì)其功能研究是否會(huì)有影響還尚未見報(bào)道。

3.4 其他研究方法

此外，研究人員還利用RNA pulldown、RIP(RNA immunoprecipitation)、CLIP (crosslinking immuno-precipitation)、PAR-CLIP (photoactivatable ribonu-cleoside-enhanced crosslinking and immunoprecipita-tion)、CLASH (cross-linking ligation and sequencing of hybrids)等分子生物學(xué)實(shí)驗(yàn)技術(shù)來分析tsRNA與核酸以及蛋白質(zhì)的相互作用(表4)，隨著對(duì)tsRNA的深入研究，創(chuàng)新更多高效的技術(shù)手段，將有利于幫助理解tsRNA的功能。

表4 tsRNA與蛋白質(zhì)/RNA相互作用的研究方法

4 結(jié)語(yǔ)與展望

早在20世紀(jì)70年代tsRNA就已經(jīng)被檢測(cè)出來，但長(zhǎng)期以來一直認(rèn)為其是tRNA隨機(jī)降解產(chǎn)物，直到最近10年，部分tsRNA的產(chǎn)生機(jī)制和生物學(xué)功能才逐漸被發(fā)現(xiàn)。tsRNA通過調(diào)控轉(zhuǎn)錄本穩(wěn)定、調(diào)節(jié)基因表達(dá)等途徑在正常生物學(xué)過程和疾病發(fā)生中發(fā)揮重要作用，目前tsRNA的研究還面臨著一些問題：(1) tsRNA命名規(guī)則還沒有統(tǒng)一；(2) tsRNA產(chǎn)生機(jī)制還未研究透徹，例如i-tRF、tRF-2產(chǎn)生方式還不清楚，不同物種中tsRNA產(chǎn)生機(jī)制是否保守也有待進(jìn)行深入研究；(3)目前對(duì)其功能的研究幾乎都是利用化學(xué)合成的RNA序列，而內(nèi)源性的tsRNA具有多種RNA修飾，因此很可能忽略了RNA修飾對(duì)tsRNA功能的影響。

盡管目前對(duì)tsRNA的認(rèn)識(shí)還不完全，但它作為一種新的功能性小非編碼RNA，在生物體中有著極為重要的作用，相信在未來的研究中將揭示其更多的生物學(xué)意義。

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The function and research methods of tRNA-derived small RNAs (tsRNA)

Jianfeng Ma1,2, Mailin Gan1,2, Li Zhu1,2, Linyuan Shen1,2

tRNA-derived small RNA (tsRNA), a class of small non-coding RNAs processed from the precursor tRNA or mature tRNA, broadly exist in organisms and have features including high conservation, structural stability and tissue specificity. According to the different paths of biogenesis, tsRNA is classified into two major types, tRNA halves and tRNA-derived RNA fragments (tRFs). Many studies have revealed that the expression of tsRNA is significantly increased under a variety of cellular stress responses, and its involvement in the regulation of stress responses is conserved across different species. tsRNA plays an important role in various biological processes by regulating transcript stability, protein translation and epigenetic processes.Recent researches have shown that tsRNAhas the potential as disease biomarkers and therapeutic targets, making it the focus of biomedical research. In this review, we summarize the research on tsRNA from aspects of biogenesis, function and research methods in order to provide a reference for relevant research.

non-coding RNA;tRNA-derived fragments; tsRNA; tRFs

2021-07-16;

2021-09-17

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：31902135，31972524)和四川省科技廳基礎(chǔ)研究項(xiàng)目(編號(hào)：2021YJ0265，2021YFYZ0007，2020YFN0147)資助[Supported by the National Natural Science Foundation of China (Nos. 31902135, 31972524) and the Sichuan Science and Technology Support Program (Nos. 2021YJ0265, 2021YFYZ0007, 2020YFN0147)]

馬劍峰，在讀碩士研究生，專業(yè)方向：動(dòng)物遺傳育種。E-mail: 1098991954@qq.com

朱礪，博士，教授，研究方向：豬遺傳育種與繁殖。E-mail: zhuli7508@163.com

沈林園，博士，講師，研究方向：豬遺傳育種與繁殖。E-mail: shenlinyuan0815@163.com

10.16288/j.yczz.21-256

2021/11/24 15:44:21

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20211123.1547.002.html

(責(zé)任編委: 宋旭)