近日,，國(guó)際著名雜志The Plant Cell 在線刊登了中國(guó)科學(xué)院微生物研究所研究人員的最新研究成果“Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein”,，文章中，研究者揭示了他們?cè)诓《境聊种谱友芯糠矫娅@得的進(jìn)展,。
在植物和病毒相互作用的長(zhǎng)期過程中,，兩者之間存在著防御、反防御的復(fù)雜斗爭(zhēng)關(guān)系,。寄主的RNA沉默機(jī)制和病毒編碼沉默抑制子就是防御-反防御斗爭(zhēng)的典型,。由黃瓜花葉病毒（CMV）編碼的基因沉默抑制子2b蛋白是早在1998年就被發(fā)現(xiàn)的抑制子之一，CMV 2b盡管是一個(gè)只有111個(gè)氨基酸的小蛋白,，卻具有非常復(fù)雜的生化特性和亞細(xì)胞定位特性,，包括與RNA沉默途徑效應(yīng)復(fù)合物的關(guān)鍵因子AGO蛋白互作，與短/長(zhǎng)的dsRNA結(jié)合,，靶向細(xì)胞核和核仁,；CMV 2b還具有復(fù)雜的抑制子活性，包括抑制轉(zhuǎn)錄后基因沉默（PTGS）和依賴RNA的DNA甲基化（RdDM）,，抑制沉默的細(xì)胞間傳遞,，抑制AGO蛋白的剪切活性,，抑制寄主的RDR6-依賴的抗病毒途徑等。但是關(guān)于2b的這111個(gè)氨基酸究竟是如何決定的2b的這些生化特性,，又是如何影響2b復(fù)雜的抑制子活性，其機(jī)制目前還研究得很不透徹,。
在國(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目,，國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃（973計(jì)劃）等項(xiàng)目資助下，中國(guó)科學(xué)院微生物研究所植物基因組學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室郭惠珊課題組,，通過分段克隆構(gòu)建了CMV 2b蛋白的11個(gè)突變體,，詳細(xì)分析了2b各種突變體的亞細(xì)胞定位、RNA結(jié)合特性,、與AGO間的互作,、對(duì)PTGS和RdDM途徑的抑制作用等。研究發(fā)現(xiàn),，2b的dsRNA的結(jié)合活性以及2b的核定位所需要的結(jié)構(gòu)域與2b與AGO蛋白的互作所需要的結(jié)構(gòu)域是分開的,。他們鑒定出2b的核仁定位信號(hào)（NoLS）位于2b結(jié)合dsRNA的結(jié)構(gòu)域（dsRBD）之間；2b和AGO蛋白的體外互作不需要dsRBD,。有趣的是,，SD2b與AGO蛋白在體內(nèi)的互作需要這個(gè)有功能的NoLS，并且2b和AGO的體內(nèi)互作改變2b和AGO的細(xì)胞核定位,。通過體內(nèi)抑制子活性檢測(cè)發(fā)現(xiàn),，2b蛋白通過捕獲siRNAs、長(zhǎng)的dsRNAs前體dsRNA,，而不是通過作用于AGO蛋白,，來(lái)實(shí)現(xiàn)其抑制子活性。也就是說(shuō),，體內(nèi)抑制PTGS和RdDM都不依賴于2b-AGO的相互作用,。（生物谷Bioon.com）
doi:10.1105/tpc.111.092718
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Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein[W]
Cheng-Guo Duana,1, Yuan-Yuan Fanga,1, Bang-Jun Zhoua,b, Jian-Hua Zhaoa,b, Wei-Na Houa, Hui Zhua,b, Shou-Wei Dingc and Hui-Shan Guoa,2
Unique among the known plant and animal viral suppressors of RNA silencing, the 2b protein interacts directly with both small interfering RNA (siRNA) and ARGONAUTE1 (AGO1) and AGO4 proteins and is targeted to the nucleolus. However, it is largely unknown which regions of the 111-residue 2b protein determine these biochemical properties and how they contribute to its diverse silencing suppressor activities. Here, we identified a functional nucleolar localization signal encoded within the 61–amino acid N-terminal double-stranded RNA (dsRNA) binding domain (dsRBD) that exhibited high affinity for short and long dsRNA. However, physical interaction of 2b with AGOs required an essential 33-residue region C-terminal to the dsRBD and was sufficient to inhibit the in vitro AGO1 Slicer activity independently of its dsRNA binding activities. Furthermore, the direct 2b–AGO interaction was not essential for the 2b suppression of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) in vivo. Lastly, we found that the 2b–AGO interactions in vivo also required the nucleolar targeting of 2b and had the potential to redistribute both the 2b and AGO proteins in nucleus. These findings together suggest that 2b may suppress PTGS and RdDM in vivo by binding and sequestering siRNA and the long dsRNA precursor in a process that is facilitated by its interactions with AGOs in the nucleolus.
