Rpa49 (originally named RPA51; systematic name SPAC2F3.03c) is the fission-yeast member of the A49/PAF53/POLR1E family and one of the 14 subunits of RNA polymerase I (Pol I), the nucleolar enzyme that transcribes the ribosomal DNA repeats into the 35S pre-rRNA. It co-purifies with Pol I, including in stationary phase, and is a non-catalytic accessory subunit: Pol I lacking it keeps non-specific polymerase activity but cannot transcribe rDNA at full rate. The gene is dispensable at high temperature but needed for growth at lower temperatures. The C-terminal two-thirds of the protein carry its function, and the protein can replace budding-yeast Rpa49 in vivo. In budding yeast, the N-terminal region of A49 forms a TFIIF-like heterodimer with A34.5. That module docks on the polymerase lobe and stimulates the RNA cleavage catalysed by A12.2. The C-terminal tandem winged-helix domain of A49 is TFIIE-like and binds DNA. Through these two parts A49 helps recruit and later release the initiation factor Rrn3 and supports processive elongation. These roles are expected to be shared by the fission-yeast protein, which belongs to the same single-subfamily PANTHER family (PTHR14440) and has the same complementing activity.
Existing Annotations Review
GO Term
Evidence
Action
Reason
GO:0001188 RNA polymerase I preinitiation complex assembly
IBA GO_REF:0000033
ACCEPT
Summary: PAINT inference that the A49-family subunit takes part in assembling the Pol I initiation complex; supported by ortholog data and cross-species complementation.
Reason: PTHR14440 is a single-subfamily family, and both S. pombe Rpa49 and the experimentally studied S. cerevisiae Rpa49 and mouse PAF53 sit beneath the PAINT node PTN000374345. In budding yeast, rpa49 deletion and C-terminal truncation partly impair recruitment of the initiation factor Rrn3 to the rDNA promoter (PMID:18086878). The A49 C-terminal tandem winged-helix domain is a TFIIE-related DNA-binding module that prefers the upstream promoter (PMID:20797630). So the subunit contributes structurally to the initiation-competent Pol I complex rather than being a bystander, which meets the participation bar. The fission-yeast protein complements an S. cerevisiae rpa49 deletion (PMID:12893961), which places it inside the clade that inherited the function. No S. pombe initiation assay exists, so this stays a phylogenetic inference, but no evidence of divergence argues against it.
The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE.
rpa51+ is required for cell growth at lower but not at higher temperatures and is able to complement S. cerevisiae rpa49Delta mutation, indicating that RPA51 is a functionally-conserved subunit of Pol I between the budding yeast and the fission yeast.
Summary: InterPro2GO DNA binding for the A49 family; supported by the budding-yeast A49 tandem winged-helix DNA-binding data.
Reason: The budding-yeast A49 C-terminal tWH domain binds DNA directly (PMID:20797630). The target matches the A49 family signatures (IPR009668, PF06870, PTHR14440). Deletion mapping of the S. pombe protein shows that its C-terminal two-thirds, the region corresponding to the tWH-bearing part of A49, is enough for function (PMID:12893961). Plain DNA binding is as specific as the evidence allows. The tWH shows only a strand and position preference, not sequence-specific promoter recognition, so a sequence-specific child term would over-state it. Nobody has measured DNA binding by the S. pombe protein directly; the claim rests on family membership.
The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE.
HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ...
MODIFY
Summary: Nuclear localization from the genome-wide YFP-ORFeome screen; correct but less specific than the nucleolar localization from the same screen.
Reason: The localization is correct: a Pol I subunit works at the rDNA in the nucleolus. The same high-throughput study gives the more specific nucleolus (GO:0005730), which this gene already carries, and nucleolar residence fits the Pol I subunit role (UniProt subcellular location Nucleus, nucleolus). Under the refinement rule the general term is replaced by nucleolus rather than kept as non-core.
we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein.
HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ...
ACCEPT
Summary: Nucleolar localization of YFP-tagged Rpa49 from the genome-wide ORFeome screen.
Reason: The nucleolus is where Pol I transcribes rDNA, and this subunit co-purifies with Pol I (PMID:12893961, PMID:33536435). The high-throughput localization is consistent with that, and UniProt independently assigns a nucleolar location by similarity. The cached record is abstract-only, so the individual image could not be inspected; we defer to PomBase's curation.
we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein.
Summary: UniProt SubCell mapping to nucleolus; concordant with the HDA row and Pol I membership.
Reason: This is the same claim as the HDA nucleolus row, here from the UniProt by-similarity subcellular-location statement. It is consistent with Pol I residence at nucleolar rDNA.
Summary: PAINT inference of Pol I complex membership, matching the target's own IDA.
Reason: Pol I membership is the defining property of the A49/PAF53/POLR1E family (PTHR14440, node PTN000374345). The target's own experimental annotation (PomBase:SPAC2F3.03c) is one of the descendant evidences behind the IBD, as expected. Fission-yeast RPA51 co-purifies with Pol I (PMID:12893961). The A49/A34.5 subcomplex was also detected by SDS-PAGE and mass spectrometry in purified S. pombe Pol I used for cryo-EM (PMID:33536435).
We also found that RPA51 associates significantly with Pol I in the stationary phase, suggesting that Pol I inactivation in the stationary phase of yeast does not result from the RPA51 dissociation.
The presence of the A49/A34.5 subunit-complex in Pol I preparations was confirmed by Coomassie−stained SDS-PAGE (Supplementary Fig. 1a) and mass-spectrometry analysis.
IDA PMID:12893961 The fission yeast RPA51 is a functional homolog of the buddi...
ACCEPT
Summary: Direct biochemical evidence that RPA51/Rpa49 is a Pol I subunit in fission yeast.
Reason: Nakagawa et al. found the protein as a Pol I subunit that stays associated with the enzyme even in stationary phase (PMID:12893961). Later cryo-EM work on purified S. pombe Pol I confirmed that the A49/A34.5 heterodimer is present in the 14-subunit enzyme. Its density is not resolved, which reflects the flexibility of the subcomplex (PMID:33536435).
We designate the corresponding protein RPA51 and gene encoding it rpa51+ since the gene encodes a Pol I subunit and an apparent molecular mass of the protein is 51 kDa.
We also found that RPA51 associates significantly with Pol I in the stationary phase, suggesting that Pol I inactivation in the stationary phase of yeast does not result from the RPA51 dissociation.
The presence of the A49/A34.5 subunit-complex in Pol I preparations was confirmed by Coomassie−stained SDS-PAGE (Supplementary Fig. 1a) and mass-spectrometry analysis.
Summary: Correct but very general InterPro2GO process term; the gene already carries the specific Pol I transcription terms.
Reason: Rpa49 takes part in transcription, but only as a Pol I subunit. The specific supported descendants are transcription elongation by RNA polymerase I (GO:0006362; S. pombe IDA plus IBA) and RNA polymerase I preinitiation complex assembly (GO:0001188; IBA). Both are already on the gene and both are is_a/part_of descendants of GO:0006351 (verified via OLS hierarchicalAncestors). Both are listed because the A49 subunit acts at initiation (Rrn3 recruitment) and at elongation (Rrn3 release, processivity, cleavage stimulation).
Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription.
These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.
GO:0006362 transcription elongation by RNA polymerase I
IBA GO_REF:0000033
ACCEPT
Summary: PAINT inference of a role in Pol I elongation, concordant with the target's IDA and with budding-yeast A49 data.
Reason: Budding-yeast A49 supports elongation in several ways. The A49/A34.5 dimerization module stimulates the intrinsic RNA cleavage of the polymerase (PMID:20797630). A49 is needed to release Rrn3 from the elongating enzyme (PMID:18086878), and rpa49 mutants lose polymerase occupancy on rDNA and are sensitive to 6-azauracil and mycophenolate, typical of elongation defects (PMID:18086878). The S. pombe protein is needed for maximal rDNA transcription in vivo and in vitro (PMID:12893961). The target's own IDA is one of the IBD's descendant evidences, so it is not circular. The subunit supports the polymerase and does not itself synthesise RNA.
Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription.
The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core.
These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.
GO:0006362 transcription elongation by RNA polymerase I
IDA PMID:12893961 The fission yeast RPA51 is a functional homolog of the buddi...
ACCEPT
Summary: S. pombe in vivo and in vitro transcript analysis showing that RPA51 is needed for maximal rDNA transcription by Pol I.
Reason: The abstract shows that RPA51 maximises specific rDNA transcription but is dispensable for non-specific transcription by Pol I. The cached record is abstract-only, so we cannot see which assays led the PomBase curator to choose elongation rather than initiation. The choice matches the budding-yeast A49 elongation phenotypes (PMID:18086878, PMID:20797630), so we defer to the curator.
Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription.
Core Functions
Non-catalytic, Pol I-specific accessory subunit that contributes to RNA polymerase I activity at nucleolar rDNA and is needed for maximal rRNA transcription, acting in initiation complex formation and elongation.
Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription.
The presence of the A49/A34.5 subunit-complex in Pol I preparations was confirmed by Coomassie−stained SDS-PAGE (Supplementary Fig. 1a) and mass-spectrometry analysis.
DNA binding through the C-terminal A49-family tandem winged-helix region, inferred from the budding-yeast ortholog within PTHR14440.
The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE.
Q: Does the S. pombe Rpa49 C-terminal region bind upstream promoter DNA with the same nontemplate-strand preference reported for the S. cerevisiae A49 tWH domain?
Suggested Experiments
Experiment: Express the S. pombe Rpa49 C-terminal region (the two-thirds sufficient for function) and measure binding to rDNA promoter and bubble substrates by EMSA or fluorescence anisotropy, comparing it with the S. cerevisiae A49 tWH.
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.
Both ProtNLM2 GO predictions describe established biology of this non-catalytic Pol I subunit, and both are less specific than supported curated annotations on the gene (LSP); RNA polymerase I complex (IDA, IBA) and transcription elongation by RNA polymerase I (IDA, IBA).
Review rationale: S. pombe RPA51/Rpa49 co-purifies with Pol I, including in stationary phase (PMID:12893961), and the A49/A34.5 subcomplex is present in purified S. pombe Pol I used for cryo-EM (PMID:33536435). Membership of a DNA-directed RNA polymerase complex is therefore supported. The gene already carries RNA polymerase I complex (GO:0005736, IDA and IBA), an is_a descendant of the predicted term, so the prediction is LSP. The protein is an accessory subunit, not a catalytic one.
Supporting Evidence:
PMID:12893961: "We also found that RPA51 associates significantly with Pol I in the stationary phase, suggesting that Pol I inactivation in the stationary phase of yeast does not result from the RPA51 dissociation."
PMID:33536435: "The presence of the A49/A34.5 subunit-complex in Pol I preparations was confirmed by Coomassie−stained SDS-PAGE (Supplementary Fig. 1a) and mass-spectrometry analysis."
Review rationale: S. pombe RPA51/Rpa49 is a Pol I subunit needed for maximal rDNA transcription in vivo and in vitro (PMID:12893961), so participation in DNA-templated transcription is supported. The exact term is already on the gene as an InterPro2GO IEA, which the gene review modifies to its specific descendants; the gene carries transcription elongation by RNA polymerase I (GO:0006362, IDA and IBA) and RNA polymerase I preinitiation complex assembly (GO:0001188, IBA). Because the prediction is materially less specific than these supported annotations, it is LSP rather than CNN.
Supporting Evidence:
PMID:12893961: "Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription."