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.
Highlights
Ribosomes are molecular machines that are essential for protein synthesis
The N-terminal domain (NTD) exhibits the SAM-dependent MTase fold, which is reminiscent of the Rossmann fold, with a central seven-stranded b sheet flanked by three a helices on each side (Fig. 1C)
We show that lack of such a catalytic activity of human dimethyladenosine transferase 1 (DIMT1) significantly decreases protein synthesis and translation fidelity, this catalytic role of DIMT1 is not required for ribosome small subunit assembly
Summary
Ribosomes are molecular machines that are essential for protein synthesis. In eukaryotes, ribosome assembly requires four ribosomal RNAs (18S, 5.8S, 25S, and 5S) [1,2,3,4] as well as ;200 indispensable assembly factors. Previous studies suggested that the expression, but not the catalytic activity, of DIMT1 is important for rRNA processing and ribosome biogenesis [19]. The only study to investigate the mechanism by which human DIMT1 influences 18S rRNA processing showed that the catalytic activity of DIMT1 is not required for this process, leaving the function of the evolutionarily conserved rRNA modification m26,6A uncharacterized [19]. Whereas the finding of a noncatalytic structural role for DIMT1 is highly credible, no evidence suggests that the catalytic role of DIMT1 is required for ribosome biogenesis or protein translation [10, 25,26,27,28,29]. F, sequencealignment of humanDIMT1 andKsgA showing an additional a-helix in human DIMT1
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