Funk Holly M, DiVita Daisy J, Sizemore Hannah E, Wehrle Kendal, Miller Catherine L W, Fraley Morgan E, Mullins Alex K, Guy Adrian R, Phizicky Eric M, Guy Michael P
Department of Chemistry & Biochemistry, Northern Kentucky University, Highland Heights, Kentucky 41076, United States.
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, United States.
ACS Omega. 2022 Apr 13;7(16):13667-13675. doi: 10.1021/acsomega.1c07231. eCollection 2022 Apr 26.
Posttranscriptional tRNA modifications are essential for proper gene expression, and defects in the enzymes that perform tRNA modifications are associated with numerous human disorders. Throughout eukaryotes, 2'--methylation of residues 32 and 34 of the anticodon loop of tRNA is important for proper translation, and in humans, a lack of these modifications results in non-syndromic X-linked intellectual disability. In yeast, the methyltransferase Trm7 forms a complex with Trm732 to 2'--methylate tRNA residue 32 and with Trm734 to 2'--methylate tRNA residue 34. Trm732 and Trm734 are required for the methylation activity of Trm7, but the role of these auxiliary proteins is not clear. Additionally, Trm732 and Trm734 homologs are implicated in biological processes not directly related to translation, suggesting that these proteins may have additional cellular functions. To identify critical amino acids in Trm732, we generated variants and tested their ability to function in yeast cells. We identified a conserved RRSAGLP motif in the conserved DUF2428 domain of Trm732 that is required for tRNA modification activity by both yeast Trm732 and its human homolog, THADA. The identification of Trm732 variants that lack tRNA modification activity will help to determine if other biological functions ascribed to Trm732 and THADA are directly due to tRNA modification or to secondary effects due to other functions of these proteins.
转录后tRNA修饰对于正确的基因表达至关重要，而负责tRNA修饰的酶的缺陷与多种人类疾病相关。在整个真核生物中，tRNA反密码子环第32位和第34位残基的2'-甲基化对于正确翻译很重要，在人类中，缺乏这些修饰会导致非综合征性X连锁智力障碍。在酵母中，甲基转移酶Trm7与Trm732形成复合物，使tRNA第32位残基发生2'-甲基化，与Trm734形成复合物，使tRNA第34位残基发生2'-甲基化。Trm732和Trm734是Trm7甲基化活性所必需的，但这些辅助蛋白的作用尚不清楚。此外，Trm732和Trm734的同源物与并非直接与翻译相关的生物学过程有关，这表明这些蛋白可能具有其他细胞功能。为了鉴定Trm732中的关键氨基酸，我们构建了变体并测试了它们在酵母细胞中的功能能力。我们在Trm732保守的DUF2428结构域中鉴定出一个保守的RRSAGLP基序，酵母Trm732及其人类同源物THADA的tRNA修饰活性都需要该基序。缺乏tRNA修饰活性的Trm732变体的鉴定将有助于确定归因于Trm732和THADA的其他生物学功能是直接由于tRNA修饰，还是由于这些蛋白的其他功能产生的次级效应。
