Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA.
Cell Rep. 2013 Nov 27;5(4):1070-81. doi: 10.1016/j.celrep.2013.10.017. Epub 2013 Nov 14.
During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.
在 miRNA 生物发生过程中，微处理器复合物（MC），其最小组成部分为 Drosha，一种 RNase III 酶，和 DGCR8，一种双链 RNA 结合蛋白，切割初级 miRNA（pri-miRNA）以释放前 miRNA 茎环结构。使用磷酸化蛋白质组学，我们在昆虫或哺乳动物细胞中表达全长人 DGCR8 时绘制了 23 个磷酸化位点。DGCR8 可以被有丝分裂 ERK/MAPK 磷酸化，表明 DGCR8 磷酸化可能对外界信号做出反应并进行整合。磷酸化 DGCR8 或磷酸酶抑制剂的表达增加了 DGCR8 和 Drosha 蛋白的细胞水平。磷酸化 DGCR8 水平的增加不是由于更高的 mRNA 水平、DGCR8 定位改变或 DGCR8 自我缔合的能力，而是由于蛋白稳定性的增加。包含磷酸化突变体或磷酸化 DGCR8 的 MC 在特定的加工活性中没有改变。然而，表达磷酸化 DGCR8 的 HeLa 细胞表现出促生长 miRNA 表达谱，并与表达磷酸化突变体 DGCR8 的细胞相比，表现出更高的增殖和划痕闭合率。
