Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (PAR-CLIP). Homo sapiens

We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Overall design: Please see individual series. In short, for AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.

本研究报道，AUF1可调控全局mRNA稳定性与翻译进程，进而促进DNA完整性的维持。整体实验设计详见各系列数据集。简言之：针对AUF1光活性增强核糖核蛋白交联免疫沉淀测序（PAR-CLIP）实验：将带有Flag标签表位（Flag epitope）的4种AUF1蛋白亚型（p37、p40、p42及p45）在HEK293细胞中过表达。总RNA测序（total RNA-Seq）实验：将HEK293细胞分别转染阴性对照小干扰RNA（siRNA）、AUF1靶向siRNA、空载体以及携带Flag标签的AUF1 p37、p40、p42、p45亚型；同时收集处于群体倍增水平（PDL）15和55代的WI-38细胞，将其分别转染阴性对照siRNA、AUF1靶向siRNA及HuR靶向siRNA。核糖体印记测序（Ribo-Seq）实验：将HeLa细胞分别转染阴性对照siRNA、AUF1靶向siRNA及HuR靶向siRNA。