Function of arginine methylation in post-transcriptional gene regulation

精氨酸甲基化通过蛋白质-蛋白质相互作用影响细胞过程。到目前为止，它在转录后调控基因表达中的作用还不是很清楚。拟议的项目将加深对蛋白质修饰有助于翻译调控的机制的理解。重点是精氨酸甲基化对多域蛋白hnRNP K功能的影响。hnRNP K是网织红细胞15-脂氧合酶(R15-LOX)mRNA翻译的特异性调节因子，也是c-Src激酶的激活剂和底物。HnRNP K在体外和体内都被甲基化，但甲基化的甲基转移酶(PRMT)、底物精氨酸和功能结果(S)尚不清楚。我在体内和体外鉴定了PRMT1为hnRNP K甲基化酶，并证明内源性hnRNP K和PRMT1共免疫共沉淀。在共转染hnRNP K、c-Src和PRMT1的HeLa细胞中，hnRNP K/c-Src相互作用和hnRNP K酪氨酸磷酸化显著降低。这支持了我的假设，即hnRNP K甲基化是一种重要的功能修饰。现在我将确定底物精氨酸并生成工具来研究hnRNP K的甲基化是否影响其在R15-LOX mRNA翻译控制中的功能及其细胞定位。

Arginine methylation affects cellular processes through protein-protein interactions. Its role in the posttranscriptional control of gene expression is ill explored so far. The proposed project will deepen the understanding of the mechanisms by which protein modifications contribute to translational regulation. The focus is on the impact of arginine methylation on the function of the multidomain protein hnRNP K. hnRNP K is a specific regulator of reticulocyte 15-lipoxygenase (r15-LOX) mRNA translation, and an activator and substrate of c-Src kinase. HnRNP K is methylated in vitro and in vivo, but the methyltransferase (PRMT), substrate arginines and functional consequence(s) of the methylation are not known. I identified PRMT1 as the hnRNP K-methylating enzyme in vitro as well as in vivo and showed that endogenous hnRNP K and PRMT1 co-immunoprecipitate. In HeLa cells co-transfected with hnRNP K, c-Src and PRMT1, the hnRNP K/c-Src interaction and hnRNP K tyrosine phosphorylation were strongly reduced. This sup ports my hypothesis that hnRNP K methylation is a functionally important modification. Now I will identify the substrate arginines and generate tools to study whether methylation of hnRNP K affects its function in r15-LOX mRNA translation control and its cellular localization.