Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery (RNA-Seq). Mus musculus

Protein Arginine MethylTransferase 5 (PRMT5) is known to mediate epigenetic control on chromatin and to functionally regulate components of the splicing machinery. In this study we show that selective deletion of PRMT5 in different organs leads to cell cycle arrest and apoptosis. At the molecular level, PRMT5 depletion results in reduced methylation of Sm proteins, aberrant constitutive splicing and in the Alternative Splicing (AS) of specific mRNAs. We identify Mdm4 as one of these mRNAs, which due to its weak 5’-Donor site, acts as a sensor of splicing defects and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in PRMT5 conditional knockout mice. Our data demonstrate a key role of PRMT5, together with p53, as guardians of the transcriptome. This will have fundamental implications in our understanding of PRMT5 activity, both in physiological conditions, as well as pathological conditions, including cancer and neurological diseases. Overall design: Total RNA was extracted from control and Prmt5 depleted Neural Stem/Progenitors Cells (NPCs) and Mouse Embryonic Fibroblasts (MEFs). Prmt5 depleted cells were treated with 4-OHT 24 hours before splitting to induce PRMT5 knockout and final libraries were sequenced in triplicates on Illumina HiSeq 2000.

蛋白质精氨酸甲基转移酶5（PRMT5）已知可介导染色质的表观遗传调控，并在功能上调控剪接机器的组分。本研究证实，在不同器官中选择性敲除PRMT5会引发细胞周期阻滞与细胞凋亡。在分子层面，PRMT5表达缺失会导致Sm蛋白甲基化水平降低、组成型剪接异常，以及特定信使RNA（mRNA）的可变剪接（Alternative Splicing，AS）。本研究鉴定出Mdm4即为这类mRNA之一：由于其5’剪接供体位点（5’-Donor site）序列较弱，Mdm4可作为剪接缺陷的传感器，并通过传导信号激活p53应答通路，为PRMT5条件性敲除小鼠中观察到的表型提供了机制层面的解释。本研究数据证实，PRMT5与p53共同作为转录组的守护者。该发现对于我们理解PRMT5在生理及病理（包括癌症与神经系统疾病）状态下的活性具有重要意义。实验设计概况：从对照组与Prmt5缺失的神经干细胞/前体细胞（Neural Stem/Progenitors Cells，NPCs）及小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts，MEFs）中提取总RNA。Prmt5缺失的细胞在传代前24小时用4-OHT处理以诱导PRMT5敲除，最终构建的文库在Illumina HiSeq 2000平台上进行三次重复测序。