The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

To uncover exon junction complex (EJC) deposition sites on cellular mRNAs, RNA footprints of EJC immuo-purified from HEK293 cells were deep sequenced. The analysis of these data revealed that major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions (-24 position) and that the majority of exon junctions carry an EJC. Unexpectedly, we find that many sites further upstream of -24 position are also enriched in these EJC footprints. These "non-canonical" sites are binding sites of EJC-interacting proteins with a subset being occupied by SR proteins. Thus, an EJC-SR protein nexus exists within spliced mRNPs and is revealed here. Overall design: Deep sequencing based profiling of EJC RNA footprints obtained by tandem RNA immunoprecipitation (RIPiT) of RNase I digested RNA:protein complexes.

为揭示细胞mRNA上的外显子连接复合体（exon junction complex, EJC）沉积位点，研究人员对从HEK293细胞中免疫纯化得到的EJC的RNA足迹进行了深度测序。对上述数据的分析显示，体内主要的"经典"EJC占据位点位于外显子接头上游24个核苷酸处（-24位），且大多数外显子接头均带有EJC。出乎意料的是，本研究发现-24位上游更远端的诸多位点同样富集此类EJC RNA足迹。这些"非经典"位点是与EJC相互作用的蛋白结合位点，其中一部分子集会被SR蛋白占据。由此可见，剪接后的信使核糖核蛋白颗粒（mRNPs）中存在EJC-SR蛋白互作复合体，本研究对此进行了阐明。整体实验设计：通过对经核糖核酸酶I（RNase I）酶解的RNA-蛋白复合物实施串联RNA免疫沉淀（tandem RNA immunoprecipitation, RIPiT）获取EJC RNA足迹，进而采用深度测序技术开展谱分析。