Perreault Audrey, Gascon Suzanne, D'Amours Annie, Aletta John M, Bachand Francois
RNA Group, Department of Biochemistry, Université de Sherbrooke, Québec J1H 5N4, Canada.
J Biol Chem. 2009 May 29;284(22):15026-37. doi: 10.1074/jbc.M109.004812. Epub 2009 Apr 9.
Schizosaccharomyces pombe Rmt3 is a member of the protein-arginine methyltransferase (PRMT) family and is the homolog of human PRMT3. We previously characterized Rmt3 as a ribosomal protein methyltransferase based on the identification of the 40 S Rps2 (ribosomal protein S2) as a substrate of Rmt3. RMT3-null cells produce nonmethylated Rps2 and show mis-regulation of the 40 S/60 S ribosomal subunit ratio due to a small subunit deficit. For this study, we have generated a series of RMT3 alleles that express various amino acid substitutions to characterize the functional domains of Rmt3 in Rps2 binding, Rps2 arginine methylation, and small ribosomal subunit production. Notably, catalytically inactive versions of Rmt3 restored the ribosomal subunit imbalance detected in RMT3-null cells. Consistent with a methyltransferase-independent function for Rmt3 in small ribosomal subunit production, the expression of an Rps2 variant in which the identified methylarginine residues were substituted with lysines showed normal levels of 40 S subunit. Importantly, substitutions within the zinc finger domain of Rmt3 that abolished Rps2 binding did not rescue the 40 S ribosomal subunit deficit of RMT3-null cells. Our findings suggest that the Rmt3-Rps2 interaction, rather than Rps2 methylation, is important for the function of Rmt3 in the regulation of small ribosomal subunit production.
粟酒裂殖酵母Rmt3是蛋白质精氨酸甲基转移酶（PRMT）家族的成员，是人类PRMT3的同源物。我们之前基于将40S核糖体蛋白S2（Rps2）鉴定为Rmt3的底物，将Rmt3表征为核糖体蛋白甲基转移酶。缺乏RMT3的细胞产生未甲基化的Rps2，并由于小亚基缺陷而表现出40S/60S核糖体亚基比例的失调。在本研究中，我们生成了一系列表达各种氨基酸取代的RMT3等位基因，以表征Rmt3在Rps2结合、Rps2精氨酸甲基化和小核糖体亚基产生中的功能域。值得注意的是，催化无活性的Rmt3版本恢复了在缺乏RMT3的细胞中检测到的核糖体亚基失衡。与Rmt3在小核糖体亚基产生中具有不依赖甲基转移酶的功能一致，一种Rps2变体的表达显示40S亚基水平正常，其中已鉴定的甲基精氨酸残基被赖氨酸取代。重要的是，Rmt3锌指结构域内消除Rps2结合的取代不能挽救缺乏RMT3的细胞的40S核糖体亚基缺陷。我们的研究结果表明，Rmt3与Rps2的相互作用而非Rps2甲基化对于Rmt3在调节小核糖体亚基产生中的功能很重要。
