RNA解螺旋酶DDX3所結合之mRNAs的鑑定與特性分析

Abstract

在真核細胞中， RNA 解螺旋酶調控著許多與 RNA 相關的代謝過程，包括 pre-mRNA 剪接 (pre-mRNA splicing) 、 mRNA 核外運輸 (mRNA export) 、核醣體生合成作用 (ribosome biogenesis)、 RNA 降解 (RNA decay) 以及蛋白轉譯 (translation) 。本實驗室在研究 mRNA 核外運輸因子 (mRNA export factor) TAP 時，發現它可能與一 DEAD-box RNA 解螺旋酶 DDX3 結合，由於 DDX3 可能參與 mRNA 的核外運輸或其他 mRNA 代謝過程，所以我們利用 mRNA 分示法搜尋可能與 DDX3 相結合的 mRNAs ，初步顯示有十個 mRNA candidates ，經由免疫沉澱實驗與 RT-PCR ，發現其中的 FBX22 以及 SHINC-2 mRNAs 可以與 DDX3 以及其相關運輸複合體蛋白結合。此外，我們也發現 DDX3 上的 SAT 區域能夠影響它對 mRNAs 的結合能力。當真核細胞遭受到環境壓力 (stress) 時，會在細胞質中形成 stress granules ，並將 48S 轉譯啟始複合體累積其中，使蛋白轉譯作用受到抑制，我們發現 DDX3 在遭受環境壓力時，會進入 stress granules ，但它與 mRNA ligands 的結合會變弱。我們亦研究 DDX3 是否參與蛋白轉譯作用中的起始步驟，初步結果顯示當大量表現 DDX3 蛋白時，轉譯啟始複合體的組合似乎會受到影響，DDX3 之 mRNA ligands 與 polyribosome 之結合亦變弱，因此 DDX3 可能抑制其 mRNA ligands 的蛋白轉譯作用。

RNA helicases are involved in many aspects of RNA metabolism including pre-mRNA splicing and mRNA export, decay and translation. We recently found that the mRNA export receptor, TAP, interacted with RNA helicase DDX3, consistence with the role of DDX3 orthologs in mRNA export and translation. To study the function of human DDX3 in mRNA biogenesis, we set out to search for its mRNA targets by using the immunoprecipitation-differential display strategy. We obtained 10 mRNA candidates and found that two of them, FBX22 and SHINC-2 mRNAs, were associated with DDX3 but, however, also bound to other mRNA export-related proteins. We further found that the helicase SAT domain of DDX3 was required for its association with mRNAs. Under cell stress conditions, DDX3 was targeted to the cytoplasmic stress granules (SGs) (Lai and Tarn, unpublished data), and its association with mRNAs was also disrupted. To examine whether DDX3 participates in translation control, we characterized translation complex assembly in the presence of overexpressed DDX3. The results showed that overexpression of DDX3 could interfere with the association of its mRNA ligands with polyribosomes. Therefore, DDX3 might play a role in translation inhibition.