NCU11362 encodes the Neurospora crassa ortholog of METTL16, a nuclear S-adenosylmethionine-dependent RNA methyltransferase of the METTL16/RlmF family. By orthology with fission yeast Mtl16 and human METTL16, it is expected to install N6-methyladenosine in the conserved ACAGAGA box of U6 spliceosomal snRNA, the sequence that base-pairs with the 5' splice site of pre-mRNA introns. In fission yeast this U6 modification is needed for efficient splicing of a subset of introns. The 541-residue protein has a predicted disordered N-terminal extension, then a methyltransferase domain that retains the catalytic NPPF motif, then a C-terminal region aligned to that of fission yeast Mtl16. Methylation of mRNA targets, as reported for animal METTL16 (for example regulation of SAM synthetase transcripts), has not been shown for any fungal ortholog. No experiments have been reported on the Neurospora protein itself.
Summary: The U6 m6A installed by METTL16 sits in the ACAGA box that pairs with the 5' splice site, and its loss in fission yeast causes retention of a subset of introns; splicing is the process this enzyme serves.
Reason: PAINT placed this term at PTN000333540, the eukaryotic METTL16 node, seeded by fission yeast Mtl16 (PomBase IGI). NCU11362 descends from that node, and the alignment to Mtl16 covers 99.5% of the yeast protein with the catalytic NPPF motif at the aligned position (file:NEUCR/NCU11362/NCU11362-bioinformatics/RESULTS-rereview.md). The m6A in U6 contacts the intron during 5' splice site recognition, so the enzyme contributes a functional modification of a spliceosome component rather than acting only upstream. No target-specific evidence of loss.
Summary: mRNA methylation by METTL16 is documented in animals (human MAT2A, worm SAM synthetase pre-mRNA), whereas the only characterised fungal ortholog, fission yeast Mtl16, has no detectable mRNA target; whether the Neurospora protein methylates any mRNA is unknown.
Reason: PAINT placed this activity at PTN000333540, the eukaryotic METTL16 node, which is a considered judgement that mRNA methylation is ancestral to the clade; NCU11362 descends from that node. The donors are all animal (human METTL16, mouse Mettl16, worm METT-10), and fission yeast Mtl16, the experimentally characterised fungal member, was not used as a donor for this term. In S. pombe m6A is undetectable in poly(A)+ RNA and U6 is the sole Mtl16 target, so the property has been lost or never deployed in at least one fungal lineage. S. pombe (Taphrinomycotina) is not the target, however, and there are no Neurospora (Pezizomycotina) data on mRNA m6A or on METTL16-type hairpins in its SAM synthetase transcript. Neither the PAINT inference nor a fungus-wide loss can be confirmed from the available evidence, so the row is left undecided rather than demoted. It is not treated as a core activity; the supported activity is GO:0120048.
Propagation Review
Root cause:UNRESOLVED
Sources checked:
PANTHER:PTN000333540UNRESOLVED
Eukaryotic METTL16 node (descendants include human, mouse, worm, Arabidopsis FIO1 and S. pombe Mtl16). mRNA methylation is supported in animal descendants and absent from the fission yeast descendant; its status in Pezizomycotina has not been tested.
UniProtKB:Q86W50UNRESOLVED
Human METTL16 methylates MAT2A mRNA hairpins; sound for the source, transfer to a fungus untested.
MGI:MGI:1914743UNRESOLVED
Mouse Mettl16; same vertebrate MAT2A-regulation context.
WB:WBGene00014228UNRESOLVED
Worm METT-10 methylates SAM synthetase pre-mRNA 3' splice sites; animal substrate, transfer to a fungus untested.
we chose S. pombe as a model organism because m6A was undetectable in the poly(A)+ RNA fraction (Supplementary Fig. 2) and the S. pombe MAT2a homolog sam1 contains no hairpin structures harboring the specific nonamer sequence targeted by METTL1669. Consequently, U6 snRNA m6A is the sole target for S. pombe Mtl16.
Summary: METTL16 orthologs from plants and animals are nuclear, and the U6 snRNA substrate is modified in the nucleus.
Reason: Nuclear localisation is supported by experiments on donors spanning plants and animals at the eukaryotic METTL16 node, and it fits the U6 substrate, which is methylated in the nucleus alongside early U6 biogenesis factors. This is a phylogenetic inference, not a Neurospora localisation experiment.
Summary: Correct but too general; the specific U6 snRNA adenine-N6 methyltransferase activity is supported and already carried by this gene.
Reason: IPR010286 (METTL16/RlmF) and PANTHER PTHR13393:SF0 both span bacterial 23S rRNA RlmF, METTL16 and the small-molecule methyltransferase PsiM, so family membership alone supports only generic methyltransferase activity. The METTL16-specific PAINT node and the conserved NPPF catalytic motif support the more specific GO:0120048, which the gene already carries by IBA.
The N6-methyladenosine (m6A) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing.
Catalytic NPPF motif: A7UX10 has a single NPPF at residues 282-285
GO:0010608 post-transcriptional regulation of gene expression
IBA GO_REF:0000033
UNDECIDED
Summary: The donor evidence is animal METTL16 regulation of SAM synthetase transcripts; whether this regulatory deployment exists in Neurospora is unknown, and the characterised fission yeast ortholog lacks the target structures.
Reason: The donors are the same animal set as for GO:0001734 (human, mouse, worm), and their basis is SAM-responsive regulation of MAT2A or sams transcripts. PAINT did not use fission yeast Mtl16 as a donor here, and the S. pombe sam1 transcript lacks the METTL16-targeted hairpins. No Neurospora data address SAM synthetase regulation by NCU11362. The splicing contribution of U6 m6A, the supported fungal role, is captured more precisely by GO:0000398. Left undecided for the same reason as GO:0001734, and not treated as core.
Propagation Review
Root cause:UNRESOLVED
Sources checked:
PANTHER:PTN000333540UNRESOLVED
SAM-synthetase mRNA regulation is documented for animal descendants; the fission yeast descendant lacks the target structures; Pezizomycotina untested.
UniProtKB:Q86W50UNRESOLVED
Human METTL16 regulates MAT2A via 3' UTR hairpins.
MGI:MGI:1914743UNRESOLVED
Mouse Mettl16; vertebrate MAT2A regulation.
WB:WBGene00014228UNRESOLVED
Worm METT-10 regulation of sams pre-mRNA splicing.
Summary: U6 snRNA ACAGA-box adenosine N6-methylation is the conserved METTL16 activity, shown directly for the fission yeast and human orthologs; this is the core activity of NCU11362.
Reason: PAINT placed this activity at the eukaryotic METTL16 node, with fission yeast, human and worm donors, so it is ancestral to fungi and inherited by NCU11362. Fission yeast Mtl16 has knockout, in vitro reconstitution and structural support for the reaction. NCU11362 aligns over the full Mtl16 sequence and retains the NPPF catalytic motif. The label's position 43 is the human numbering; the homologous adenosine is A37 in S. pombe, and the Neurospora position has not been mapped.
The N6-methyladenosine (m6A) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing.
The catalytic NPPF motif is retained at the aligned position.
Core Functions
U6 snRNA adenosine N6-methyltransferase. Uses S-adenosylmethionine to methylate the adenosine in the ACAGAGA box of U6 snRNA in the nucleus; the modified U6 supports efficient 5' splice site recognition during pre-mRNA splicing. Inferred from the eukaryotic METTL16 PAINT node and fission yeast Mtl16 experiments.
The N6-methyladenosine (m6A) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing.
Q: Does Neurospora crassa have any METTL16-dependent m6A outside U6 snRNA (for example on SAM synthetase transcripts), or is U6 the sole target as in fission yeast?
Suggested Experiments
Experiment: Delete NCU11362 and measure U6 snRNA m6A by RNA mass spectrometry, poly(A)+ RNA m6A levels, and intron retention by RNA-seq, compared with wild type.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
NCU11362 is a METTL16-family RNA N6-adenosine methyltransferase inferred to modify U6 snRNA using S-adenosylmethionine. The conserved U6 modification promotes accurate pre-mRNA splice-site recognition. U6 modification and its splicing role are inferred from the eukaryotic METTL16 PAINT node and fission yeast Mtl16 experiments; whether the Neurospora protein also methylates mRNA is unresolved.
Review rationale: Primary metabolic process is correct but far less specific than the gene's existing annotation to mRNA splicing, via spliceosome (GO:0000398), an IBA from the eukaryotic METTL16 PAINT node (PTN000333540) that the gene review accepts; GO:0000398 is a descendant of GO:0044238 via RNA processing and RNA metabolic process. The underlying support is the conserved METTL16 U6 snRNA m6A activity shown experimentally for fission yeast Mtl16, which NCU11362 aligns to over its full length with the catalytic NPPF motif retained. PANTHER PTHR13393:SF0 alone would not justify this, because it also contains bacterial RlmF and PsiM; the transfer rests on the METTL16 PAINT node.
Supporting Evidence:
PMID:40841561: "The N6-methyladenosine (m6A) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing."
PMID:34050143: "We found that a subset of introns was retained in mRNAs, indicating a splicing defect caused by loss of m6A in U6 snRNA."