線蟲體內DEAD-box解旋酶和微小核糖核酸let-7基因間交互作用分析

Abstract

目前，DEAD-box家族中的RNA解旋酶已知會參與在許多改變RNA結構或RNA和蛋白之間交互作用的過程中，但是對於它們在microRNA生成或作用的過程中所扮演的角色還不是很清楚，而線蟲在基因分析實驗及RNA干擾實驗操作上的優點，讓我們決定用它作為我們RNA干擾掃描的生物模式，我們希望能藉此找到會參與在microRNA作用或生成過程中的DEAD-box解旋酶。目前對於線蟲的let-7 microRNA和其vulva爆裂性狀、seam cell發育缺陷以及退化的成蟲基因表現等異時性性狀的調控關係，大家已經很了解，並且成功地將其運用在許多篩選microRNA調控因子或作用目標的實驗上，因此首先我們找出線蟲帶有DEAD-box保守序列的蛋白後，希望藉由RNA干擾實驗將它們的基因表現抑制後同樣地觀察成蟲表現性狀，從這些蛋白中篩選出會參與在microRNA作用或功能中的DEAD-box RNA解旋酶。我們先對線蟲65個DEAD-box RNA解旋酶中的48個進行篩選，並且藉由觀察異時性性狀篩選到七個DEAD-box解旋酶：F01F1.7、cgh-1、 vbh-1、 F58E10.3、 mog-4、 H27M09.1、 Y65B4A.6，我們在這些DEAD-box解旋酶的RNA干擾實驗中，觀察到不同程度的let-7功能缺失的性狀表現，這可能代表它們在let-7 microRNA的生成或功能表現中扮演著一定的角色。接著，我們選出幾個let-7突變品系，這些突變品系包括let-7表現較弱的let-7(mg279)、對溫度有高度敏感的let-7(n2853)、Argonaute全剔除品系alg-1(gk214)以及剔除兩個let-7 microRNA家族的品系mir-48; mir-241(nDf51)，在選出這些品系中我們進行找到的七個DEAD-box解旋酶的RNA干擾實驗，觀察這些DEAD-box解旋酶和let-7生成或功能的突變之間的基因交互作用，結果我們在這些交互作用中觀察到了不同程度的性狀加成作用。我們更進一步觀察到F01F1.7, F58E10.3, mog-4 and Y65B4A.6的RNA干擾實驗中let-7表現量有降低的現象，代表這四個DEAD-box解旋酶確實有可能參與在microRNA的生成過程之中。另外，我們成功地構築一個可穩定表現帶有HA標記的F58E10.3的線蟲品系，亦得到F58E10.3多株抗體，我們會繼續將其用於未來對於F58E10.3和let-7 microRNA之間關係的生化方法研究上。

RNA helicases of the DEAD-box protein family have been shown to participate in many pathways that are associated with RNA structural conformation changes and RNA-protein interaction but their roles in microRNA processing and function remain unclear. Here we show an RNAi screen in the model organism C. elegans, taking the advantages of C. elegans genetics analysis and RNA interference tools, for DEAD-box RNA helicases that are involved in microRNA processing and/or function. First we searched for the proteins containing conserved sequences of DEAD-box helicases in C. elegans and then executed RNA interference to knock down their coding genes for reverse genetics analysis. We focused on the heterochronic phenotypes of the let-7 microRNA, such as vulva bursting, seam cell developmental defects and retarded adult gene expression, since the phenotypes are clear and have been used in several successful screens of microRNA regulators or targets in previous studies. We have screened 48 of 65 DEAD-box RNA helicases in C. elegans and found seven DEAD-box helicases: F01F1.7, cgh-1, vbh-1, F58E10.3, mog-4, H27M09.1, Y65B4A.6, whose RNAi knock-down caused let-7 loss-of-function phenotypes, may play a role in let-7 function. Analysis of genetic interactions of these DEAD-box RNA helicases and several alleles directing weak to moderate let-7 mutant phenotypes, including a weak allele let-7(mg279), a temperature-sensitive allele let-7(n2853), a null Argonaute mutant alg-1(gk214) and a deletion of two let-7 family miRNAs mir-48; mir-241(nDf51), have shown synergistic severities of heterochronic phenotypes in different levels. Moreover, reduced expression of F01F1.7, F58E10.3, mog-4 and Y65B4A.6 resulted in a decrease of the let-7 miRNA, suggesting a role of these DEAD-box RNA helicases in microRNA biogenesis. We have raised a rabbit polyclonal antibody against F58E10.3 and successfully expressed a HA-tagged F58E10.3 in C. elegans for future studies on the role of F58E10.3 in let-7 biogenesis and/or function by biochemical approaches.