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The U6 snRNA m6A Methyltransferase METTL16 is required for accurate and efficient splicing of Drosophila pre-mRNAs

ABSTRACT: In this study, we generated viable mettl16 knockout Drosophila and GFP knock-in Drosophila via the CRISPR/Cas9 technique, which provides an excellent in vivo model for investigating the precise functions of the Mettl16 protein. Subsequent staining assays revealed that Drosophila Mettl16 is a ubiquitously expressed nuclear protein, and its depletion does not affect the translational level in flies. Loss of Mettl16 results in multiple developmental phenotypes, including developmental delay, reduced locomotor activity, flightlessness, and sterility. Through MeRIP-seq and splicing analyses, we found that Mettl16 mediates m6A methylation of only a small subset of mRNAs. The reduced m6A modification level on its conserved methylation substrate U6 snRNA induces significant global changes in splicing events

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE324097 | GEO | 2026/07/17

REPOSITORIES: GEO

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The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retention

Project description:Maintenance of the intracellular levels of the methyl donor S-adenosylmethionine (SAM) is essential for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of MAT2A, which encodes the only SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3´ UTR. Increasing METTL16 occupancy on the MAT2A 3´ UTR is sufficient to induce efficient splicing. We propose that under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which in turn promotes MAT2A splicing. Interestingly, human and S. pombe METTL16 methylate the U6 spliceosomal snRNA at a sequence identical to hp1. These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

2017-05-18 | GSE90914 | GEO

METTL16 is a N6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs

Project description:N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16-interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5’ splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.

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Functional study of m6A methyltransferase METTL16 in drosophila development

Project description:N6-methyladenosine (m6A) is one of the most prevalent RNA modifications in eukaryotes. The METTL16 enzyme catalyzes the m6A modification at position 43 of U6 snRNA, which is very important for the 5'-splice site recognition by U6 during pre-mRNA splicing. Loss of METTL16 results in the death of mouse early embryos, making it difficult to study the function of METTL16 in development. Here we characterize METTL16 in the context of Drosophila melanogaster development. We showed that dMETTL16 localizes in nuclei of all endoreduplicating cells, such as cells in salivary glands, with a pattern coinciding with the replication timing. In addition, dMETTL16 is present in diploid cells of the gonad, suggesting that it plays a role in gonadal development. To further determine the function of dMETTL16, we used the CRISPR/Cas9 system to construct dMETTL16 mutations and discovered that mutants are semilethal at the larval stage while surviving adults are female sterile. More interestingly, a 50% reduction of U6 gene dosage leads to complete lethality of dMETTL16 mutants at the larval stage suggesting that loss of U6 function is one of the primary defects in the mutants. Using a novel antibody-based in vitro methylation assay, we proved that U6 is a substrate of dMETTL16. To determine whether the phenotypes depend on m6A methylase activity, we constructed point mutations of the active site of dMETTL16, and discovered that ovarian development was restored while maternal lethality ensures. These results suggest that dMETTL16 has important functions that are independent of its methyltransferase activity. Our study establishes Drosophila as a genetically tractable model for the study of dMETTL16’s function and can be explored to uncover additional dMETTL16 m6A targets.

2026-07-05 | GSE271111 | GEO

RNA Methyltransferase METTL16’s Protein Domains Have Differential Functional Effects on Cell Processes

Project description:METTL16, a human m6A RNA methyltransferase, contains multiple RNA binding domains and is known to modify U6 and MAT2A RNAs. Usingmutagensis, we generated HEK293 cell lines stabling expressing nutated or WT forms of METTL16 to dtermine the imapct these mutations have on cell processes after removal of endogenous METTL16. We performed bottom-up, untargeted data dependent proteomics analysison all five cell lines to determine the global changes in peptide/protein abdundances; we identified 200-300 statistically significant altered proteinscompared to the wild-type exogenous METTL16 clone.

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The catalytic efficiency of METTL16 affects cellular methyltransferase activities by governing the intracellular S-adenosylmethionine setpoint

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Methylation of structured RNA by the m6A writer METTL16 is essential for mouse embryonic development

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2023-01-12 | GSE190044 | GEO

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Molecular insights into THUMPD2-mediated m2G methylation of the U6 snRNA

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2026-05-25 | PXD053510 | Pride

Molecular insights into THUMPD2-mediated m2G methylation of the U6 snRNA

Project description:MMARY THUMPD2 installs N2-methylguanosine (m2G) at position 72 of the U6 snRNA, and this modification, which lies at the catalytic core of the spliceosome, is required for efficient splicing of weak splice sites. However, when during the U6 lifecycle this modification is installed and what features of the U6 snRNA and THUMPD2 are required for methylation of G72 remain elusive. Here, we show that U6 associated with THUMPD2 is not oligouridylated and that late-acting U6 snRNP biogenesis factors are enriched with THUMPD2. We map RNA crosslinking sites within the N-terminal region and C-terminal Rossman-fold methyltransferase domain of THUMPD2, and show the requirement of these domains for THUMPD2 interaction with U6 in cells. Using a newly developed m2G-sensitive deoxyribozyme to monitor U6-m2G72 levels in cellular RNAs, we demonstrate the requirement of SAM binding and interaction with the THUMPD2 cofactor TRMT112 for efficient methylation. Our data further reveal that 2’-O-methylation of C62, C63 and A70 do not increase the affinity of THUMPD2 for the U6 internal stem-loop (ISL), but enhance m2G methylation of G72. Nucleotide substitutions within the loop region of the ISL that extend the stem similarly increase m2G72 installation, demonstrating that the stability of the ISL is key for methylation by THUMPD2. Together these results provide a new layer of understanding of how an RNA modification that fine-tunes pre-mRNA splicing is introduced.

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