PROTEIN ARGININE METHYLATION AND RNA MATURATION

本研究的总体目标是了解蛋白质精氨酸甲基化的生理作用及其对酿酒酵母mRNA成熟的影响。鉴于这种翻译后修饰在RNA代谢酶中的普遍存在，它可能在这一过程中发挥重要作用。在进一步探索这一假设时，第一个目标将是分析精氨酸甲基化对Hrp 1p的影响，Hrp 1p是一种必需的酵母蛋白，是一类被称为异质核糖核蛋白或hnRNPs的蛋白质的成员。为了提供一个基础的分析，二甲基精氨酸残基在Hrp 1p将首先绘制。接下来，我们将分析这种修饰对三种Hrp 1p活性的影响；1) mRNA 3'端加工，2)rna结合，3)核质穿梭。第二个目的是通过确定过程的可逆性来检验蛋白质精氨酸甲基化是否代表一种信号机制。如果是这样，计划是识别和表征相应的去甲基化酶。第三个目标是利用生物化学和分子生物学方法的结合来鉴定和表征与蛋白质精氨酸甲基转移酶（Hmt1p）物理相互作用或功能重叠的蛋白质。作为这一目标的一部分，接受这种修饰的细胞蛋白将被编目。目的是了解这些蛋白质在细胞中的定位，以及它们是否具有共同的序列和细胞活动。由于精氨酸甲基转移酶的结构从酵母到人类细胞已经很好地保守，因此遗传上可适应的酵母酿酒酵母代表了这些研究的理想模式生物。最近的数据表明，一些人类精氨酸甲基化蛋白与分子疾病（即FMR1：脆性X智力迟钝综合征）有关。此外，在病毒RNA成熟酶中存在甲基化精氨酸意味着这种修饰在病毒mRNA加工（即单纯疱疹ICP27）中起作用。此外，人精氨酸甲基化hnRNPs参与影响其他细胞过程的RNA代谢。更好地了解精氨酸甲基化不仅可以增强对细胞RNA成熟的理解，还可以为细胞感染过程中RNA成熟的病毒机制提供见解。

The overall goal of the proposed research is to understand the physiological role of protein arginine methylation and its impact on the maturation of mRNA in the yeast Saccharomyces cerevisiae. Given the prevalence of this post-translational modification among RNA metabolism enzymes, it is likely to play an important role in the process. In exploring this hypothesis further, the first aim will be to analyze the impact of arginine methylation on Hrp 1p, an essential yeast protein that is a member of a class of proteins termed heterogeneous nuclear ribonucleoproteins or hnRNPs. To provide a foundation for this analysis, the dimethylarginine residues within Hrp 1p will first be mapped. Next, the effect of this modification on three Hrp 1p activities will be assayed; l) mRNA 3'-end processing, 2) RNA-binding, and 3) nucleocytoplasmic shuttling. The second aim examines whether protein arginine methylation represents a signaling mechanism by determining the reversibility of the process. If so, the plan is to identify and characterize the corresponding demethylase. The third aim utilizes a combination of biochemical and molecular biological approaches to identify and characterize proteins that physically interact or functionally overlap with the protein arginine methyltransferase enzyme (Hmt1p). As part of this aim, cellular proteins that receive this modification will be cataloged. The objective is to learn where these proteins are localized in the cell, and whether they share common sequences and cellular activities. Since the structures of arginine methyltransferases have been well conserved from yeast to human cells, the genetically amenable yeast Saccharomyces cerevisiae represents an ideal model organism for these studies. Recent data implicates some human arginine methylated proteins in molecular diseases (i.e. FMR1: Fragile X Mental Retardation Syndrome). Additionally, the presence of methylated arginines among viral RNA maturation enzymes implies a role for this modification in viral mRNA processing (i.e. herpes simplex ICP27). Furthermore, human arginine methylated hnRNPs participate RNA metabolism which impacts additional cellular processes. A better understanding of arginine methylation should not only enhance the understanding of cellular RNA maturation, but also provide insights into viral mechanisms for RNA maturation during cell infection.