Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands.
Nucleic Acids Res. 2018 Nov 30;46(21):11592-11604. doi: 10.1093/nar/gky861.
RNA G-quadruplexes (rG4s) are secondary structures in mRNAs known to influence RNA post-transcriptional mechanisms thereby impacting neurodegenerative disease and cancer. A detailed knowledge of rG4-protein interactions is vital to understand rG4 function. Herein, we describe a systematic affinity proteomics approach that identified 80 high-confidence interactors that assemble on the rG4 located in the 5'-untranslated region (UTR) of the NRAS oncogene. Novel rG4 interactors included DDX3X, DDX5, DDX17, GRSF1 and NSUN5. The majority of identified proteins contained a glycine-arginine (GAR) domain and notably GAR-domain mutation in DDX3X and DDX17 abrogated rG4 binding. Identification of DDX3X targets by transcriptome-wide individual-nucleotide resolution UV-crosslinking and affinity enrichment (iCLAE) revealed a striking association with 5'-UTR rG4-containing transcripts which was reduced upon GAR-domain mutation. Our work highlights hitherto unrecognized features of rG4 structure-protein interactions that highlight new roles of rG4 structures in mRNA post-transcriptional control.
RNA 四链体(rG4s)是 mRNA 中的二级结构,已知其影响 RNA 转录后机制,从而影响神经退行性疾病和癌症。详细了解 rG4-蛋白相互作用对于理解 rG4 功能至关重要。在此,我们描述了一种系统的亲和蛋白质组学方法,该方法鉴定了 80 种高可信度的相互作用蛋白,这些蛋白组装在 NRAS 癌基因 5'非翻译区(UTR)中的 rG4 上。新型 rG4 相互作用蛋白包括 DDX3X、DDX5、DDX17、GRSF1 和 NSUN5。鉴定出的大多数蛋白质都含有甘氨酸-精氨酸(GAR)结构域,值得注意的是,DDX3X 和 DDX17 中的 GAR 结构域突变会破坏 rG4 的结合。通过全转录组单核苷酸分辨率 UV 交联和亲和富集(iCLAE)鉴定 DDX3X 的靶标,发现与 5'-UTR 中含有 rG4 的转录本存在惊人的关联,而 GAR 结构域突变则降低了这种关联。我们的工作强调了 rG4 结构-蛋白相互作用的先前未被认识到的特征,突出了 rG4 结构在 mRNA 转录后调控中的新作用。