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N6 -methyladenosine (m 6 A) is the most abundant modification on messenger RNAs (mRNAs) and is catalyzed by methyltransferase-like protein 3 (Mettl3). To understand the role of m 6 A in a self-renewing somatic tissue, we deleted Mettl3 in epidermal progenitors in vivo. Mice lacking Mettl3 demonstrate marked features of dysfunctional development and self-renewal, including a loss of hair follicle morphogenesis and impaired cell adhesion and polarity associated with oral ulcerations. We show that Mettl3 promotes the m 6 A-mediated degradation of mRNAs encoding critical histone modifying enzymes. Depletion of Mettl3 results in the loss of m 6 A on these mRNAs and increases their expression and associated modifications, resulting in widespread gene expression abnormalities that mirror the gross phenotypic abnormalities. Collectively, these results have identified an additional layer of gene regulation within epithelial tissues, revealing an essential role for m 6 A in the regulation of chromatin modifiers, and underscoring a critical role for Mettl3-catalyzed m 6 A in proper epithelial development and self-renewal.

Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18 S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region., (© 2023 Gonskikh et al.; Published by Cold Spring Harbor Laboratory Press.)

Dimethyladenosine transferase 1 (DIMT1) is an evolutionarily conserved RNA N 6,6 -dimethyladenosine (m 2 6,6 A) methyltransferase. DIMT1 plays an important role in ribosome biogenesis, and the catalytic activity of DIMT1 is indispensable for cell viability and protein synthesis. A few RNA-modifying enzymes can install the same modification in multiple RNA species. However, whether DIMT1 can work on RNA species other than 18S rRNA is unclear. Here, we describe that DIMT1 generates m 2 6,6 A not only in 18S rRNA but also in small RNAs. In addition, m 2 6,6 A in small RNAs were significantly decreased in cells expressing catalytically inactive DIMT1 variants (E85A or NLPY variants) compared with cells expressing wildtype DIMT1. Both E85A and NLPY DIMT1 variant cells present decreased protein synthesis and cell viability. Furthermore, we observed that DIMT1 is highly expressed in human cancers, including acute myeloid leukemia. Our data suggest that downregulation of DIMT1 in acute myeloid leukemia cells leads to a decreased m 2 6,6 A level in small RNAs. Together, these data suggest that DIMT1 not only installs m 2 6,6 A in 18S rRNA but also generates m 2 6,6 A-containing small RNAs, both of which potentially contribute to the impact of DIMT1 on cell viability and gene expression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)

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 18 S rRNA N 6,6 -dimethyladenosine (m 2 6,6 A) 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 m 2 6,6 A levels in 18 S rRNA. Expression of a catalytically inactive variant, DIMT1-E85A, in WT and DIMT1 +/- cells significantly decreased m 2 6,6 A levels in 18S rRNA, indicating a dominant-negative effect of this variant on m 2 6,6 A 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 m 2 6,6 A in 18 S rRNA, is essential for 40S assembly., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Shen et al.)

The posttranscriptional modification of messenger RNA (mRNA) and transfer RNA (tRNA) provides an additional layer of regulatory complexity during gene expression. Here, we show that a tRNA methyltransferase, TRMT10A, interacts with an mRNA demethylase FTO (ALKBH9), both in vitro and inside cells. TRMT10A installs N 1 -methylguanosine (m 1 G) in tRNA, and FTO performs demethylation on N 6 -methyladenosine (m 6 A) and N 6 ,2'- O -dimethyladenosine (m 6 A m ) in mRNA. We show that TRMT10A ablation not only leads to decreased m 1 G in tRNA but also significantly increases m 6 A levels in mRNA. Cross-linking and immunoprecipitation, followed by high-throughput sequencing results show that TRMT10A shares a significant overlap of associated mRNAs with FTO, and these mRNAs have accelerated decay rates potentially through the regulation by a specific m 6 A reader, YTHDF2. Furthermore, transcripts with increased m 6 A upon TRMT10A ablation contain an overrepresentation of m 1 G9-containing tRNAs codons read by tRNA Gln(TTG) , tRNA Arg(CCG) , and tRNA Thr(CGT) These findings collectively reveal the presence of coordinated mRNA and tRNA methylations and demonstrate a mechanism for regulating gene expression through the interactions between mRNA and tRNA modifying enzymes., Competing Interests: The authors declare no competing interest.