Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.
RNA. 2010 Jul;16(7):1317-27. doi: 10.1261/rna.2057810. Epub 2010 May 19.
The N(1)-methyl-Adenosine (m(1)A58) modification at the conserved nucleotide 58 in the TPsiC loop is present in most eukaryotic tRNAs. In yeast, m(1)A58 modification is essential for viability because it is required for the stability of the initiator-tRNA(Met). However, m(1)A58 modification is not required for the stability of several other tRNAs in yeast. This differential m(1)A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m(1)A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m(1)A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA(Met) is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m(1)A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m(1)A58 hypomodified tRNAs. Most m(1)A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m(1)A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.
在大多数真核 tRNA 中，TPsiC 环中的保守核苷酸 58 上的 N(1)-甲基腺苷（m(1)A58）修饰存在。在酵母中，m(1)A58 修饰对于生存是必需的，因为它是起始 tRNA(Met)稳定性所必需的。然而，m(1)A58 修饰对于酵母中几种其他 tRNA 的稳定性不是必需的。这种不同的 m(1)A58 对不同 tRNA 物种的反应提出了一个问题，即在正常细胞中，某些 tRNA 是否在 A58 处被低甲基化，以及 A58 处的低甲基化如何影响 tRNA 的稳定性和功能。在这里，我们应用基因组方法来确定人细胞系中 m(1)A58 低甲基化 tRNA 的存在，并展示 A58 低甲基化如何影响 tRNA 的稳定性和参与翻译。我们基于微阵列的方法基于引物延伸的允许性来检测 m(1)A58 低甲基化 tRNA 物种的存在。在所检查的五个人类细胞系中，大约四分之一的所有 tRNA 物种以不同的量被低甲基化，并且低甲基化 tRNA 的模式非常相似。在所有情况下，都没有检测到低甲基化的起始 tRNA(Met)，这与该修饰在稳定人细胞中的这种 tRNA 的要求一致。m(1)A58-甲基转移酶的亚基的 siRNA 敲低导致生长缓慢表型，并显著增加 m(1)A58 低甲基化 tRNA 的量。大多数 m(1)A58 低甲基化 tRNA 可以与多核糖体以不同程度结合。我们的结果显示了人 tRNA 中 m(1)A58 低甲基化的明显模式，并且与这样一种观点一致，即该修饰在不同的环境中微调 tRNA 的功能。
