Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada.
Cell Rep. 2020 May 26;31(8):107693. doi: 10.1016/j.celrep.2020.107693.
The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.
哺乳动物的 mRNA 核输出过程被认为通过核糖核蛋白重塑在核孔复合体的细胞质侧终止。我们对人类 RNA 结合 E3 泛素连接酶的物理相互作用进行了严格的亲和纯化质谱筛选。由此产生的蛋白质相互作用网络揭示了 RNA 结合 E3 泛素连接酶 MKRN2 与 GLE1 之间的相互作用，GLE1 是一种 DEAD 盒解旋酶激活因子，与 mRNA 输出终止有关。我们在斑马鱼模型中评估了 MKRN2 与 GLE1 的上位性。通过 MO 介导的敲低或基于 CRISPR/Cas9 的 MKRN2 敲除部分挽救了 GLE1 耗竭时观察到的视网膜发育缺陷，这与 GLE1 和 MKRN2 之间的功能关联一致。通过核糖体组学方法，我们表明 MKRN2 选择性地结合到多种 mRNA 的 3'UTR 上，并且 MKRN2 相关 mRNA 的核输出在 MKRN2 敲低时增强。总之，我们认为 MKRN2 与 GLE1 相互作用，选择性地上调 mRNA 的核输出和视网膜发育。
