【摘要】：RNA干擾(RNA interference, RNAi)是一類真核生物中保守的調(diào)控基因表達(dá)的機(jī)制。RNAi中的核心組分是small RNAs (sRNAs)和Argonaute (AGO)家族蛋白。sRNAs同AGO蛋白結(jié)合形成RNA-induced silencing complexes (RISCs)。RISCs在sRNAs的介導(dǎo)下通過堿基互補(bǔ)配對的方式在轉(zhuǎn)錄水平(Transcriptional gene silencing, TGS)或者轉(zhuǎn)錄后水平(Post-transcriptional gene silencing, PTGS)特異調(diào)節(jié)基因的表達(dá)。在擬南芥中,根據(jù)sRNAs的生成和作用機(jī)制的不同,將其分為四類,包括microRNAs (miRNAs), trans-acting sRNAs (ta-siRNAs), natural antisense siRNAs (na-siRNAs)和heterochromatin siRNAs (hc-siRNAs)。miRNAs是一類長度為21 nt的sRNAs,它在植物的多種生物學(xué)過程中起著重要的作用。miRNAs的表達(dá)具有組織和發(fā)育的特異性。在擬南芥中,miRNAs基因由RNA聚合酶Ⅱ(RNA polymerase Ⅱ, Pol Ⅱ)轉(zhuǎn)錄得到初級miRNAs (primary miRNAs, pri-miRNAs), pri-miRNAs具有一個發(fā)卡結(jié)構(gòu)。RNA結(jié)合蛋白DAWDLE (DDL)可以結(jié)合和穩(wěn)定pri-miRNAs,并且招募Dicer-like 1 (DCL1)去識別和切割pri-miRNAs。DCL1在雙鏈RNA結(jié)合蛋白HYPONASTC LEAVES 1 (HYL1)和鋅指蛋白SERRATE (SE)的協(xié)助下,將pri-miRNAs準(zhǔn)確有效的切割成為成熟的miRNAs。DCL1, HYL1和SE在細(xì)胞核內(nèi)形成一個切割復(fù)合物,被稱為Dicing-bodies (D-bodies)。成熟的miRNAs進(jìn)入到AGO1蛋白中,通過切割靶基因的mRNA或者抑制其翻譯來調(diào)節(jié)基因的表達(dá)。為了加深我們對miRNA通路機(jī)制的理解,我們利用一個高效表達(dá)可使擬南芥表皮毛成簇的人工miRNA的轉(zhuǎn)基因系,建立了針對miRNA通路的遺傳篩選體系。通過誘變篩選,我們得到了一些miRNA活性增強(qiáng)(enhanced miRNA activity, ema)或減弱(compromised miRNA activity, cma)的突變體。我們之前報道的EMA1編碼一個importin β家族蛋白,可以負(fù)調(diào)節(jié)miRNA進(jìn)入AGO1的過程。在本研究中,我們進(jìn)一步以emal突變體為材料,進(jìn)行了emal的抑制子篩選(suppressor of emal, soe),以更敏感地獲得調(diào)節(jié)miRNA產(chǎn)生和活性的新因子。在本研究中,我們分析了其中一個突變體soe4。該突變發(fā)生在AT2G19380中。AT2G19380編碼一個包含四個鋅指結(jié)構(gòu)域和一個RNA結(jié)合結(jié)構(gòu)域(RRM)的未知功能蛋白。soe4突變中成熟miRNA和pri-miRNA的積累量均下降。SOE4定位于D-body中,且能夠與D-body組分DCL1,HYL1和SE相互作用。此外,SOE4在體外可以還結(jié)合pri-miRNA和pre-miRNA。我們的研究結(jié)果表明,SOE4在擬南芥miRNA通路中可能作為miRNA前體的結(jié)合蛋白起著穩(wěn)定pri-miRNA或者pre-miRNA的作用。
[Abstract]:RNA interference (RNA interference, RNAi) is the mechanism of conserved regulation of gene expression in eukaryotes. The core component of RNAi is the binding of small RNAs (sRNAs) and Argonaute (AGO) family proteins to AGO proteins to form RNA-induced silencing complexes (RISCs) .RISCs mediated by sRNAs. Pairings take place at the transcriptional level of (Transcriptional gene silencing, TGS) or at the post-transcriptional level (Post-transcriptional gene silencing, PTGS) specifically regulates the expression of genes). In Arabidopsis thaliana, according to the formation and mechanism of sRNAs, it is divided into four categories. MiRNAs including microRNAs (miRNAs), trans-acting sRNAs (ta-siRNAs), natural antisense siRNAs (na-siRNAs) and heterochromatin siRNAs (hc-siRNAs). MiRNAs are a class of 21 nt sRNAs, which play an important role in many biological processes of plants. The expression of miRNAs has tissue and developmental specificity. In Arabidopsis thaliana, the miRNAs gene is transcribed by RNA polymerase 鈪，

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