pcif1 encodes the zebrafish cap-specific adenosine methyltransferase (CAPAM, zCAPAM; EC 2.1.1.62), a nuclear SAM-dependent enzyme that N6-methylates the 2'-O-methyladenosine found at the first transcribed position of m7G-capped mRNAs, converting the m7GpppAm cap into m7Gpppm6Am. The protein has an N-terminal WW domain, which in the vertebrate family binds the Ser5-phosphorylated C-terminal domain of RNA polymerase II and couples cap methylation to early transcription, followed by a helical domain and a Rossmann-fold methyltransferase domain carrying the catalytic NPPF motif. The zebrafish enzyme was the construct used for the crystal structures of CAPAM bound to S-adenosyl-L-methionine and m7G-capped RNA, which defined the cap-recognition pocket and the residues required for catalysis. The m6Am cap mark has been linked to altered translation and stability of the modified transcripts, although the direction of the translational effect differs between studies.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT places nuclear localization at a CAPAM-family node (PTN001094508) seeded by human PCIF1 (Q9H4Z3, nucleus by EXP/IDA) and the fly ortholog. Zebrafish Pcif1 retains the same domain architecture, including the WW domain that binds the phosphorylated RNA polymerase II CTD, so it inherits the nuclear, co-transcriptional setting of the family. Reason: The node placement is sound: the substrate (the first transcribed nucleotide of a capped, nascent mRNA) and the interaction partner (Ser5-phosphorylated Pol II CTD) both restrict the enzyme to the nucleus, and the zebrafish protein conserves the WW domain (UniProt FT DOMAIN 43..77) and the catalytic domain that was crystallized. No zebrafish-specific imaging exists, but nothing about the zebrafish sequence argues for loss or divergence of the localization, so the inherited assertion is accepted as a core location. Supporting Evidence: file:DANRE/pcif1/pcif1-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000250|UniProtKB:Q9H4Z3} PMID:30467178 we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of the UniProt SUBCELLULAR LOCATION keyword (SL-0191, Nucleus), which itself is a by-similarity statement from human PCIF1. Reason: Same claim as the IBA row and consistent with the enzyme's nuclear substrate (the cap of nascent Pol II transcripts) and its Pol II CTD-binding WW domain. Accepted as a correct, if redundant, location. Supporting Evidence: file:DANRE/pcif1/pcif1-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000250|UniProtKB:Q9H4Z3} |
| GO:0016422 mRNA (2'-O-methyladenosine-N6-)-methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic mapping (ARBA, InterPro IPR039881, RHEA:22744, EC 2.1.1.62) to the exact reaction m7GpppAm + SAM -> m7Gpppm6Am + SAH. For this gene the mapping is not just inferred: the zebrafish protein (residues 178-673) was crystallized with SAM and m7G-capped RNA, and its catalytic activity and active-site mutants were assayed in the discovery paper. Reason: The electronic term coincides with the experimentally demonstrated activity of zebrafish CAPAM itself (see the IDA row from PMID:30467178 and the UniProt CATALYTIC ACTIVITY line with ECO:0000269 evidence). This is the core molecular function of the gene product. Supporting Evidence: file:DANRE/pcif1/pcif1-uniprot.txt Cap-specific adenosine methyltransferase that catalyzes file:DANRE/pcif1/pcif1-uniprot.txt X-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) OF 178-673 IN COMPLEX WITH PMID:30467178 The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. |
| GO:0099122 RNA polymerase II C-terminal domain binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the PCIF1 family entry (IPR039881). PCIF1 was originally named as a phosphorylated CTD-interacting factor, and the discovery paper identified CAPAM as the factor that binds the Ser5-phosphorylated Pol II CTD through its WW domain. The zebrafish protein carries the WW domain (residues 43-77). Reason: The interaction is a genuine, family-conserved property that recruits the enzyme to early transcription complexes, but it is the targeting mechanism rather than the activity that defines the gene product. It has not been tested on the zebrafish protein (the crystallized construct, 178-673, lacks the WW domain), so it is kept as a non-core, inferred molecular function alongside the core methyltransferase activity. Supporting Evidence: PMID:30467178 we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II file:DANRE/pcif1/pcif1-uniprot.txt AltName: Full=Phosphorylated CTD-interacting factor 1 |
| GO:0005634 nucleus | ISS GO_REF:0000024 | ACCEPT | Summary: Curator-judged sequence-similarity transfer from human PCIF1 (Q9H4Z3), which is nuclear by direct experimental evidence. Two GOA rows (2019 and 2024) carry the same term, evidence and source. Reason: Orthology to human PCIF1 is unambiguous (same domain organisation, and the zebrafish enzyme reproduces the human cap-specific activity in vitro), and nuclear localization follows from the nuclear substrate and Pol II association. Accepted; see the IBA row for the phylogenetic argument. Supporting Evidence: file:DANRE/pcif1/pcif1-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000250|UniProtKB:Q9H4Z3} |
| GO:0006397 mRNA processing | IDA PMID:30467178 Cap-specific terminal N (6)-methylation of RNA by an RNA pol... | ACCEPT | Summary: The discovery paper assigned mRNA processing to the zebrafish protein on the basis of its cap-specific N6-methylation of the first transcribed adenosine of capped mRNAs, demonstrated with the zebrafish construct in the structural and enzymatic work. m6Am formation is a covalent maturation step on the 5' end of Pol II transcripts. Reason: The participation test is met directly: Pcif1 catalyses the modification step itself. The term is broad, but it is the process term that UniProt curators consistently attach to CAPAM across all five human IDA papers, and the obvious narrower alternative (GO:0016556 mRNA modification) is not a descendant of mRNA processing in the current ontology, so a MODIFY would swap rather than refine. Accepted as the core process; the more specific term is raised under suggested_questions. Abstract-only cache; the zebrafish-specific assays are documented in the UniProt reference block (function, catalytic activity and mutagenesis on the zebrafish protein). Supporting Evidence: PMID:30467178 N 6, 2'-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. PMID:30467178 as a cap-specific adenosine methyltransferase (CAPAM) responsible for N 6-methylation of m6Am file:DANRE/pcif1/pcif1-uniprot.txt S-ADENOSYL-L-METHIONINE AND M7G-CAPPED RNA, FUNCTION, CATALYTIC ACTIVITY, file:DANRE/pcif1/pcif1-deep-research-falcon.md m6Am is detectable in zebrafish mRNA |
| GO:0016422 mRNA (2'-O-methyladenosine-N6-)-methyltransferase activity | IDA PMID:30467178 Cap-specific terminal N (6)-methylation of RNA by an RNA pol... | ACCEPT | Summary: Direct assay of the zebrafish enzyme. UniProt records that PMID:30467178 determined the crystal structure of zebrafish CAPAM (178-673) with SAM and m7G-capped RNA, measured its function and catalytic activity, and tested mutants of the substrate-binding and catalytic residues (Arg-239, Arg-269, Asn-558, Phe-561, Glu-563, Trp-593, Pro-596/Pro-597, His-612, Phe-619). Reason: This is the experimentally demonstrated core activity of the zebrafish gene product, matching the RHEA:22744 reaction and EC 2.1.1.62 exactly. The cached record is abstract-only, but the UniProt reference block and ECO:0000269 catalytic-activity evidence document the zebrafish assays, and the human ortholog carries the same term from five independent IDA papers. Supporting Evidence: file:DANRE/pcif1/pcif1-uniprot.txt Cap-specific adenosine methyltransferase that catalyzes file:DANRE/pcif1/pcif1-uniprot.txt S-ADENOSYL-L-METHIONINE AND M7G-CAPPED RNA, FUNCTION, CATALYTIC ACTIVITY, PMID:30467178 as a cap-specific adenosine methyltransferase (CAPAM) responsible for N 6-methylation of m6Am |
| GO:0045727 positive regulation of translation | ISS GO_REF:0000024 | MARK AS OVER ANNOTATED | Summary: Transferred from human PCIF1, where it rests on one IMP result: transcriptome-wide analysis in PCIF1-knockout cells suggested that m6Am promotes translation of the modified mRNAs. Two subsequent human studies reached different conclusions, and human PCIF1 now also carries negative regulation of translation (GO:0017148, IDA) and a NOT annotation to regulation of translation (GO:0006417, IDA). UniProt flags the disagreement in a CAUTION note. Reason: The claim is downstream of the enzyme's actual work (installing m6Am); whether that mark increases, decreases or does not change translation is contested even in the source organism, and the sign of the effect is exactly what this term asserts. Transferring a disputed directional regulatory term to zebrafish, where no translation data exist, over-states what is known. The annotation is not wrong in the sense that Pcif1 writes a mark that can influence translation, so it is marked over-annotated rather than removed. Supporting Evidence: PMID:30467178 A transcriptome-wide analysis revealed that N 6-methylation of m6Am promotes the translation of capped mRNAs. file:DANRE/pcif1/pcif1-uniprot.txt However, another study did not observe a clear file:DANRE/pcif1/pcif1-uniprot.txt report, m6A(m) inhibits mRNA translation without affecting mRNA |
| GO:0099122 RNA polymerase II C-terminal domain binding | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Sequence-similarity transfer from human PCIF1, which binds the Ser5-phosphorylated Pol II CTD by IDA (the property that gave the protein its name). The zebrafish protein conserves the WW domain responsible for this interaction. Reason: Same term and argument as the InterPro IEA row: a conserved recruitment interaction that positions the enzyme on nascent transcripts, kept as a non-core molecular function because the defining activity is the cap methyltransferase reaction and the binding has not been assayed on the zebrafish protein. Supporting Evidence: PMID:30467178 we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II file:DANRE/pcif1/pcif1-uniprot.txt /note="WW" |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:30467178 Cap-specific terminal N (6)-methylation of RNA by an RNA pol... | KEEP AS NON CORE | Summary: The zebrafish CAPAM crystal structures (PDB 6IRX, 6IRY, 6IRZ, 6IS0; 1.8-2.0 A) were solved with S-adenosyl-L-methionine bound in the methyltransferase domain, giving direct structural evidence that the zebrafish protein binds the methyl donor. Reason: SAM binding is real and directly observed for this protein, but it is the cofactor-binding component of the methyltransferase reaction rather than an independent function, so it is kept as non-core beside the accepted GO:0016422 activity that already implies it. Supporting Evidence: file:DANRE/pcif1/pcif1-uniprot.txt X-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) OF 178-673 IN COMPLEX WITH file:DANRE/pcif1/pcif1-uniprot.txt S-ADENOSYL-L-METHIONINE AND M7G-CAPPED RNA, FUNCTION, CATALYTIC ACTIVITY, PMID:30467178 The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. |
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Download this section (compressed HTML)Q: Should CAPAM/Pcif1 be annotated to GO:0016556 mRNA modification (the covalent-alteration term used for METTL3) in addition to, or instead of, the broad GO:0006397 mRNA processing that UniProt curators currently use for all PCIF1 orthologs?
Q: Is the WW-domain interaction with Ser5-phosphorylated Pol II CTD conserved in zebrafish Pcif1, and is the zebrafish enzyme, like the human one, inhibited by zinc?
Experiment: Generate a pcif1 loss-of-function zebrafish line and quantify cap m6Am by LC-MS/MS of decapped mRNA to confirm that Pcif1 is the sole m6Am writer in vivo, then profile translation (ribosome profiling) and mRNA stability to resolve the direction of the m6Am effect in a vertebrate organism.
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