探討DEAD-box核糖核酸解旋酶DDX17與OASL蛋白間交互作用

Abstract

DEAD-box核糖核酸解旋酶DDX17(p72) 屬於DEAD-box 核糖核酸解旋酶家族的一員(family)，這個家族中的成員，在相當多物種間，序列保留(DEAD-box) 具有高度相似性。DEAD-box核糖核酸解旋酶具有水解三磷酸腺苷 (ATP)，且將自行蛋白結構改變，使得核糖核酸鏈的結構重新整理或是影響其與蛋白間的交互作用。在先前的研究中，DDX17 已經被報導過參與在調控轉錄(transcription)和RNA剪接(splicing)中，例如DDX17 會與DDX5 調控肌纖維母細胞或表皮細胞的增生與分化，利用與異質核醣核酸蛋白 (hnRNP H/F) 的交互作用。此外在癌症細胞中，DDX17 也成為微小核醣核酸(microRNA)生合成的因子之一，癌症細胞的形成有關，在癌症細胞中，細胞核中的YAP 會和DDX17 鍵結，使得DDX17無法與其他因子組成 Microprocessor complex，參與在微小核醣核酸的生合成，使得在細胞中的微小核醣核酸總表現量下降。而在DDX17 抗病毒的研究中，也有發現DDX17會與病毒的核醣核酸上的莖環結構結合，使得病毒的複製受到干擾。而實驗室希望能了解更多DDX17所參與的生物路徑，利用蛋白質體的技術找到數個候選蛋白，其中也包含了2′-5′-oligoadenylate synthase-like (OASL) protein。OASL 曾經被報導參與在RIG-I 參與的免疫訊息傳遞路徑，因此我們認為先前DDX17可以與病毒的核醣核酸鏈結合，成為RNA 病毒的感應受體，或許在OASL利用與DDX17有交互作用的現象上，協助先天性免疫 (innate immunity) 的進行。OASL 是 2′-5′-oligoadenylate synthase (OAS) family的一員，OAS 家族中還包含OAS1-3的成員，這個家族除了OASL外，都具有2′-5′-oligoadenylate synthase的能力，會與病毒RNA結合，消耗三磷酸腺苷並合成數個腺苷(adenosine, A)的聚合體，這個腺苷的聚合物(2′-5′-oligoadenylates)會活化核醣核酸水解酶 (RNase L) ，將病毒RNA水解。OASL不具如此功能，但具有將RIG-I的CARD domain泛素化 (ubiquitylation)。在此篇文章，我們發現了一個可能的OASL亞型蛋白可被anti-OASL 抗體辨認，且只出現在poly(I:C) 處理後的 HeLa 細胞中，利用免疫共沉澱 (immunoprecipitation) 的方式發現其與DDX17有交互作用。

DDX17 (p72) belongs to the DEAD-box RNA helicase family, which is evolutionarily conserved from bacteria to human. The DEAD-box RNA helicase hydrolyzes ATP and then, consuming the energy, rearrange RNA structures or redirect RNA-protein interaction. Previously, DDX17 has been implicated in regulation of transcription and splicing. In recent studies, DDX17 and DDX5 have been shown to control proliferation of myoblasts and epithelial cells by regulating splicing, in cooperation with hnRNP H/F, of a subset of genes. In addition, DDX17 has been reported as a factor for biogenesis of microRNAs that are related to carcinogenesis. In cancer cells, nuclear yes-associated protein (YAP) binds to DDX17 and then interferes with the Microprocessor complex, causing global downregulation of miRNA levels. Moreover, the RNA binding ability of DDX17 for stem-loop structures makes it a putative viral RNA sensor. To better understand the functional plasticity of DDX17 and to search for its mechanistic partners, I sought to identify DDX17-interacting proteins by immunoprecipitation following biochemical approaches. Among the candidates, I chose the 2′-5′-oligoadenylate synthase-like (OASL) protein, which has been implicated in RIG-I-related autoimmunity, for further examination, focusing on DDX17-OASL interaction and its relevance in RNA virus sensing mechanisms. OASL belongs to the family of OAS proteins, including OAS1-3, which are capable to bind viral RNAs and synthesize 2′-5′-oligoadenylate (2-5A) to activates RNase L for viral RNA degradation. Different to OAS1-3, OASL does not possess the oligoadenylate synthase activity instead ubiquitination. It has been reported that OASL is involved in activating the antiviral pathway by helping RIG-I ubiquitination. Here, I show that a putative OASL isoform which could be recognized by OASL antibody and only expressed upon poly(I:C) treatment in HeLa cells, interacts with DDX17 in co-immunoprecipitation.