Department of Biochemistry, Indian Institute of Science, Bangalore, India.
University of California, Davis, CA, USA.
FEBS J. 2019 Dec;286(23):4693-4708. doi: 10.1111/febs.15057. Epub 2019 Sep 23.
The fate of messenger RNA in cytoplasm plays a crucial role in various cellular processes. However, the mechanisms that decide whether mRNA will be translated, degraded or stored remain unclear. Single stranded nucleic acid binding protein (Sbp1), an Arginine-Glycine-Glycine (RGG-motif) protein, is known to promote transition of mRNA into a repressed state by binding eukaryotic translation initiation factor 4G1 (eIF4G1) and to promote mRNA decapping, perhaps by modulation of Dcp1/2 activity. Sbp1 is known to be methylated on arginine residues in RGG-motif; however, the functional relevance of this modification in vivo remains unknown. Here, we report that Sbp1 is arginine-methylated in an hnRNP methyl transferase (Hmt1)-dependent manner and that methylation is enhanced upon glucose deprivation. Characterization of an arginine-methylation-defective (AMD) mutant provided evidence that methylation affects Sbp1 function in vivo. The AMD mutant is compromised in causing growth defect upon overexpression, and the mutant is defective in both localizing to and inducing granule formation. Importantly, the Sbp1-eIF4G1 interaction is compromised both for the AMD mutant and in the absence of Hmt1. Upon overexpression, wild-type Sbp1 increases localization of another RGG motif containing protein, Scd6 (suppressor of clathrin deficiency) to granules; however, this property of Sbp1 is compromised in the AMD mutant and in the absence of Hmt1, indicating that Sbp1 repression activity could involve other RGG-motif translation repressors. Additionally, the AMD mutant fails to increase localization of the decapping activator DEAD box helicase homolog to foci and fails to rescue the decapping defect of a dcp1-2Δski8 strain, highlighting the role of Sbp1 methylation in decapping. Taken together, these results suggest that arginine methylation modulates Sbp1 role in mRNA fate determination.
细胞质中信使 RNA 的命运在各种细胞过程中起着至关重要的作用。然而,决定 mRNA 是否被翻译、降解或储存的机制尚不清楚。单链核酸结合蛋白(Sbp1)是一种精氨酸-甘氨酸-甘氨酸(RGG 基序)蛋白,已知通过结合真核翻译起始因子 4G1(eIF4G1)促进 mRNA 进入抑制状态,并通过调节 Dcp1/2 活性促进 mRNA 脱帽。Sbp1 已知在 RGG 基序的精氨酸残基上被甲基化;然而,这种修饰在体内的功能相关性尚不清楚。在这里,我们报告 Sbp1 以 hnRNP 甲基转移酶(Hmt1)依赖的方式被精氨酸甲基化,并且在葡萄糖剥夺时甲基化增强。精氨酸甲基化缺陷(AMD)突变体的特征提供了证据表明甲基化影响 Sbp1 在体内的功能。AMD 突变体在过表达时会导致生长缺陷,并且该突变体在定位和诱导颗粒形成方面都有缺陷。重要的是,AMD 突变体和缺乏 Hmt1 都会破坏 Sbp1-eIF4G1 相互作用。在过表达时,野生型 Sbp1 增加另一种含有 RGG 基序的蛋白质 Scd6(网格蛋白缺陷抑制剂)向颗粒的定位;然而,Sbp1 的这种特性在 AMD 突变体和缺乏 Hmt1 时被削弱,表明 Sbp1 的抑制活性可能涉及其他 RGG 基序翻译抑制剂。此外,AMD 突变体未能增加去帽激活因子 DEAD 盒螺旋酶同源物向焦点的定位,并且未能挽救 dcp1-2Δski8 菌株的去帽缺陷,突出了 Sbp1 甲基化在去帽中的作用。总之,这些结果表明精氨酸甲基化调节 Sbp1 在 mRNA 命运决定中的作用。