Structural and catalytic roles of the human 18S rRNA methyltransferases DIMT1 in ribosome assembly and translation

Abstract

<jats:p>Ribosomal RNA (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 wildtype 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 wildtype 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 wildtype DIMT1, DIMT1-E85A could not revert the defects in protein translation. We found that the differences between this variant and the wildtype 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.</jats:p>

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea

“…In fact, analyses performed in the model bacterium E. coli [this study and ( 25 )] provide a very similar picture in this organism, where no major effect on protein synthesis and stability, and only discrete effects on a subset of the E. coli proteome could be scored [this study and ( 25 )]. This is in stark contrast to a recent analysis using human cell culture where a greater effect on proteostasis was observed ( 122 ). However, the use of overexpression of catalytic inactive KsgA/Dim1, in a haploinsufficient human KsgA/Dim1 background, may account for the exacerbating effect on cellular transcription/translation observed under these experimental conditions ( 122 ).…”

“…This is in stark contrast to a recent analysis using human cell culture where a greater effect on proteostasis was observed ( 122 ). However, the use of overexpression of catalytic inactive KsgA/Dim1, in a haploinsufficient human KsgA/Dim1 background, may account for the exacerbating effect on cellular transcription/translation observed under these experimental conditions ( 122 ). Indeed, overexpression of catalytic inactive KsgA/Dim1 leads to dominant-negative effects on various aspects of prokaryotic cellular physiology [( 16 ), this study see Figure 2 ].…”

“…Previous studies, suggested a role of the KsgA/Dim1-dependent modifications to ensure the functional integrity of the SSU A- and P-site ( 24 , 27 , 28 , 31 ). Accordingly, absence of bacterial KsgA/Dim1 and its activity are associated to general translation initiation defects and decrease translation fidelity ( 24 , 26 , 121 , 122 ). Whereas these functional considerations could suggest a global impact of KsgA/Dim1 loss of function on cellular proteostasis, our analysis does not provide evidence for a pleiotropic effect on translation activity associated to the absence of KsgA/Dim1 and/or its catalytic activity.…”

Regulation of translation by methylation multiplicity of 18S rRNA

“…Here, we introduced a highly conservative E85Q mutation to eliminate the methyltransferase activity of Dim1p ( Figure 1E ), but unfortunately, this E85Q mutant is still defective in SSU biogenesis ( Figures S5A and S5B ) and rRNA processing ( Figure S6B ). Recent structural work reveals that the E85A mutation has little impact on the overall conformation of human DIMT1 ( Shen et al, 2020 ), and conceivably, the E85Q mutation would be expected to be even less disruptive. Still, even such a conservative change leads to rRNA processing defects.…”

Age-Dependent and Tissue-Specific Alterations in the rDNA Clusters of the Panax ginseng C. A. Meyer Cultivated Cell Lines

“…Alterations in methylation patterns can be associated with methylase/demethylase activity; it was previously shown that the total demethylase (VaDem1 and VaDem2) expression in grape cell lines was lower than in the leaves of the native plant [ 72 ]. Although the biological functions of the rRNA modifications remain mainly unclear, alterations in the rRNA modifications occur during development, environmental changes, and disease, and dysregulation of methylation can affect ribosomal ligand binding and translation accuracy [ 73 , 74 , 75 ].…”