Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins
Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins
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DOI：
10.3390/biom5031441
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发表时间：
2015-09-01
期刊：
影响因子：
5.5
通讯作者：
Vogel, Christine
中科院分区：
文献类型：
--
作者：
Bish, Rebecca;Cuevas-Polo, Nerea;Vogel, Christine
DDX6（p54/RCK）是一种人类RNA解旋酶，在mRNA降解和翻译抑制中起核心作用。为了帮助我们理解DDX6如何执行这些多种功能，我们进行了首次无偏向的大规模研究，利用免疫沉淀和质谱法绘制以DDX6为中心的蛋白质 - 蛋白质相互作用组图谱。以DDX6为诱饵，我们确定了一组高可信度和高质量的蛋白质相互作用伙伴，它们在RNA代谢和核糖体蛋白功能方面富集。该筛选具有高度特异性，使真阳性数量最大化，正如通过已知功能和相互作用对81%（47/58）的不依赖RNA的相互作用蛋白进行验证所表明的那样。重要的是，我们通过使用基于核酸酶的消化来消除RNA，从而将间接相互作用伙伴的数量降至最低。我们描述了11种新的相互作用蛋白，包括参与剪接的蛋白质，这是DDX6一种尚未知晓的作用。我们更详细地验证和描述了DDX6与核脆性X智力低下相互作用蛋白2（NUFIP2）以及两种先前未被描述的蛋白质FAM195A和FAM195B（在此称为颗粒蛋白 - 1和颗粒蛋白 - 2，或GRAN1和GRAN2）之间的相互作用。我们表明NUFIP2、GRAN1和GRAN2不是P小体成分，但在受到应激时会重新定位到应激颗粒中，这表明它们在细胞应激反应中的翻译抑制功能。通过一项解决DDX6多种复合物成员关系的互补分析，我们进一步验证了这些相互作用伙伴以及剪接因子的存在。由于DDX6还与E3 SUMO连接酶TIF1相互作用，我们检测并观察到在DDX6的相互作用伙伴中SUMO化显著富集。我们的结果代表了对RNA生命周期和定位的关键调节因子的直接相互作用伙伴最全面的筛选，突出了新的应激颗粒成分以及可能的DDX6功能，其中许多功能可能在真核生物中是保守的。
DDX6 (p54/RCK) is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58) of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2) and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2). We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6's multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1, we tested for and observed a significant enrichment of sumoylation amongst DDX6's interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functionsmany of which are likely conserved across eukaryotes.