p38-MK2 regulate RNA metabolism after UV light-induced DNA damage

Ultraviolet (UV) light induces the formation of bulky UV photoproducts in the genome that interfere with DNA replication and transcription. It is well-established how human cells repair UV light-induced DNA lesions, however the signaling pathways and mechanisms that regulate transcription after exposure to UV light are poorly understood. Here, we provide a systematic view on dynamic protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on canonical DNA damage kinases and the p38 MAP kinase pathway. Notably, we demonstrate that p38 and its downstream effector kinase MK2 are responsible for one quarter of protein phosphorylation induced by UV light. We identify RNA binding proteins as primary targets and 14-3-3 family proteins as direct readers of UV light-induced, p38-MK2-dependent phosphorylation. Importantly, we demonstrate that UV light triggers rapid and dynamic phosphorylation of the negative elongation factor (NELF) complex subunit NELFE on serine 115 that mediates its binding to 14-3-3. NELFE interaction with 14-3-3 stabilizes NELFE and RNA pol II interaction on the chromatin and inhibits transcriptional elongation, thereby promoting cell survival after UV light. Analysis of NELFE occupancy genome-wide in U2OS cells by ChIP-seq.

紫外线（Ultraviolet，UV）可在基因组中诱导形成大体积UV光产物，此类产物会干扰DNA复制与转录过程。目前学界已明确人类细胞修复UV诱导DNA损伤的机制，但对于UV暴露后调控转录的信号通路与分子机制，仍知之甚少。本研究通过系统性分析UV诱导的蛋白质磷酸化动态谱，揭示了磷酸化事件对经典DNA损伤激酶及p38丝裂原活化蛋白激酶（p38 MAP kinase）通路的依赖关系。值得注意的是，本研究证实p38及其下游效应激酶MK2介导了UV诱导的四分之一蛋白质磷酸化事件。我们鉴定出RNA结合蛋白为该磷酸化的主要靶标，同时发现14-3-3家族蛋白是UV诱导、依赖p38-MK2通路的磷酸化的直接识别因子。重要的是，本研究证实UV可快速且动态地使负延伸因子（negative elongation factor，NELF）复合物亚基NELFE的丝氨酸115位点发生磷酸化，该修饰介导其与14-3-3蛋白的结合。NELFE与14-3-3的结合可稳定NELFE及RNA聚合酶II（RNA polymerase II，RNA pol II）在染色质上的相互作用，并抑制转录延伸，从而促进UV暴露后的细胞存活。本研究通过染色质免疫沉淀测序（chromatin immunoprecipitation sequencing，ChIP-seq）分析了U2OS细胞中NELFE的全基因组结合占据情况。