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.

中文翻译：

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人类 18S rRNA 甲基转移酶 DIMT1 在核糖体组装和翻译中的结构和催化作用。

rRNA 修饰酶参与核糖体组装。然而，这些酶的催化活性对于核糖体组装和其他细胞过程是否重要尚不完全清楚。在这里，我们报告了 WT 人二甲基腺苷转移酶 1 (DIMT1)（一种 18S rRNA N6,6-二甲基腺苷 (m26,6A) 甲基转移酶）的晶体结构，以及使用催化失活 DIMT1 变体获得的结果。我们发现 DIMT1+/- 杂合 HEK 293T 细胞的 40S 分数显着降低，蛋白质合成减少，但 18S rRNA 中的 m26,6A 水平没有重大变化。催化失活变体 DIMT1-E85A 在 WT 和 DIMT1+/- 细胞中的表达显着降低了 18S rRNA 中的 m26,6A 水平，表明该变体对 m26,6A 水平具有显性负效应。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 WT DIMT1 不同，DIMT1-E85A 无法恢复蛋白质翻译中的缺陷。我们发现该变体与 WT 酶之间的差异延伸至 DNA 损伤反应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，并且无论 18S rRNA 中 m26,6A 的催化活性如何，DIMT1 的整体蛋白质支架对于 40S 组装都是必需的。