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Abstract：
rRNA-modifying enzymes participate in ribosome assembly. However, whether the catalytic activities of these enzymes are important for the ribosome assembly and other cellular processes is not fully understood. Here, we report the crystal structure of WT human dimethyladenosine transferase 1 (DIMT1), an 18S rRNA N6,6-dimethyladenosine (m26,6A) methyltransferase, and results obtained with a catalytically inactive DIMT1 variant. We found that DIMT1+/- heterozygous HEK 293T cells have a significantly decreased 40S fraction and reduced protein synthesis but no major changes in m26,6A levels in 18S rRNA. Expression of a catalytically inactive variant, DIMT1-E85A, in WT and DIMT1+/- cells significantly decreased m26,6A levels in 18S rRNA, indicating a dominant-negative effect of this variant on m26,6A levels. However, expression of the DIMT1-E85A variant restored the defects in 40S levels. Of note, unlike WT DIMT1, DIMT1-E85A could not revert the defects in protein translation. We found that the differences between this variant and the WT enzyme extended to translation fidelity and gene expression patterns in DNA damage response pathways. These results suggest that the catalytic activity of DIMT1 is involved in protein translation and that the overall protein scaffold of DIMT1, regardless of the catalytic activity on m26,6A in 18S rRNA, is essential for 40S assembly.
摘要：
rRNA修饰酶参与核糖体组装。然而,这些酶的催化活性对核糖体组装和其他细胞过程是否重要尚不完全清楚。这里,我们报道了WT人二甲基腺苷转移酶1(DIMT1)的晶体结构，18SrRNAN6,6-二甲基腺苷（m26,6A）甲基转移酶，以及使用无催化活性的DIMT1变体获得的结果。我们发现DIMT1+/-杂合HEK293T细胞具有显著降低的40S分数和减少的蛋白质合成,但在18SrRNA中的m26,6A水平没有重大变化。表达无催化活性的变体，DIMT1-E85A,在WT和DIMT1+/-细胞中，18SrRNA中的m26,6A水平显着降低，表明该变体对m26,6A水平的显性负面影响。然而,DIMT1-E85A变体的表达恢复了40S水平的缺陷。值得注意的是，与WTDIMT1不同,DIMT1-E85A无法修复蛋白质翻译的缺陷.我们发现该变体与WT酶之间的差异扩展到DNA损伤应答途径中的翻译保真度和基因表达模式。这些结果表明，DIMT1的催化活性参与蛋白质翻译，并且DIMT1的整体蛋白质支架，无论18SrRNA对m26,6A的催化活性如何，是必不可少的40S装配。
