Affinage

RPAP2

Putative RNA polymerase II subunit B1 CTD phosphatase RPAP2 · UniProt Q8IXW5

Length
612 aa
Mass
69.5 kDa
Annotated
2026-06-10
18 papers in source corpus 17 papers cited in narrative 17 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RPAP2 (yeast ortholog Rtr1) is a regulator of RNA Polymerase II that couples the transcription cycle to the phosphorylation state of the Pol II C-terminal domain (CTD) (PMID:19394294, PMID:22137580). It associates with the active Pol II complex and dephosphorylates Ser5 (and, in dual-specificity assays, Tyr1) of the CTD, driving the transition from initiation to elongation and influencing transcription termination (PMID:19394294, PMID:22137580, PMID:24951832). In human cells RPAP2 is recruited to snRNA genes through direct recognition of the phospho-Ser7 CTD mark, where it both removes Ser5 phosphorylation and recruits the Integrator complex (PMID:22137580). Structural work places RPAP2 between the RPB1 and RPB5 jaw domains of Pol II, a position incompatible with downstream DNA engagement; through steric clashes RPAP2 blocks TFIIF binding and inhibits pre-initiation complex assembly independently of any catalytic activity, with its loss causing promoter accumulation of TFIIF and Pol II (PMID:34021257, PMID:35476980). Beyond its CTD role, RPAP2/Rtr1 contributes to Pol II biogenesis, cooperating with assembly factors Gpn3 and Npa3 to mediate Rpb1-Rpb2 and Rpb4/7 assembly—functions that are also phosphatase-independent (PMID:36190433, PMID:35216121). RPAP2 protein levels are controlled by FBXW7-mediated ubiquitylation following p38/GSK3 phosphorylation at Ser562/Thr565 and counteracted by USP7, a pathway whose disruption drives hepatic cystogenesis with RPAP2 as the causal effector (PMID:39932049). Whether RPAP2/Rtr1 possesses intrinsic catalytic phosphatase activity or acts largely as a non-catalytic cofactor remains directly contested across structural and biochemical studies (PMID:22781759, PMID:24951832, PMID:26933063).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2008 Medium

    Established that the uncharacterized factor Rtr1 is a bona fide component of the active Pol II machinery rather than a peripheral regulator, by showing physical association and genetic ties to core Pol II subunits.

    Evidence Co-IP, genetic suppressor screen, and microscopy in yeast linking Rtr1 to RPB5/RPB7/RPB9 and inducible transcription

    PMID:18408053

    Open questions at the time
    • No molecular activity assigned
    • Cytoplasmic-nuclear shuttling role unexplained
    • No human data
  2. 2009 High

    Answered what Rtr1 does to Pol II by identifying it as a CTD Ser5 phosphatase required for the Ser5-to-Ser2 transition, defining its place in the transcription cycle.

    Evidence ChIP positioning, whole-cell phosphorylation analysis, and deletion phenotyping in yeast

    PMID:19394294

    Open questions at the time
    • Intrinsic versus cofactor catalysis not resolved
    • No structural basis for activity
    • Termination defect mechanism undefined
  3. 2011 High

    Extended the model to human RPAP2 and revealed mark-directed recruitment, showing phospho-Ser7 reads RPAP2 to snRNA genes where it both removes Ser5-P and brings in Integrator.

    Evidence siRNA knockdown, ChIP, Co-IP, in vitro phosphatase assay, and Ser7Ala mutational analysis in human cells

    PMID:22137580

    Open questions at the time
    • Direct Ser7-P binding determinants not mapped structurally
    • Integrator recruitment interface unknown
  4. 2012 High

    Challenged the phosphatase model by solving an Rtr1 structure lacking any identifiable active site and failing to detect catalytic activity in vitro, raising a non-catalytic cofactor hypothesis.

    Evidence X-ray crystallography of K. lactis Rtr1 plus extensive in vitro phosphatase assays

    PMID:22781759

    Open questions at the time
    • Negative enzymatic result may reflect assay conditions
    • Does not exclude in vivo activity
    • No bound substrate
  5. 2014 High

    Reasserted intrinsic catalysis by showing autoinhibited dual-specificity (Ser5/Tyr1) activity and that an activity-reducing point mutant phenocopies deletion, directly contradicting the no-activity structural study.

    Evidence In vitro phosphatase assay, site-directed mutagenesis, and yeast complementation

    PMID:24951832

    Open questions at the time
    • Conflict with prior structural study unresolved
    • Autoinhibition relief mechanism unknown
  6. 2014 Medium

    Refined recruitment logic by showing Rtr1 preferentially binds hyperphosphorylated Pol II and depends on Ser2 kinase Ctk1 for association during elongation.

    Evidence Quantitative MS interactome and affinity purification in WT and ctk1Δ yeast

    PMID:24671508

    Open questions at the time
    • Direct Ser2-P dependence not shown biochemically
    • Single lab
  7. 2016 High

    Provided a structural active-site model—a phosphoryl transfer domain with a trapped sulfate—and tied active-site mutagenesis to loss of activity and growth, supporting a distinct conserved catalytic mechanism.

    Evidence 2.6 Å crystal structure of S. cerevisiae Rtr1 with mutagenesis, in vitro assay, and complementation

    PMID:26933063

    Open questions at the time
    • Mechanism distinct from known phosphatase families not fully defined
    • Persistent disagreement with negative structural study
  8. 2016 Medium

    Defined the genome-wide consequences of Rtr1 loss, connecting Ser5-P regulation to downstream chromatin marks via Set2-dependent H3K36me3.

    Evidence ChIP-chip of CTD phosphorylation and histone methylation in RTR1 deletion yeast

    PMID:27247267

    Open questions at the time
    • Direct versus indirect effect on Set2 not separated
    • No human validation
  9. 2020 High

    Resolved how Rtr1 shapes termination, showing it restrains the NNS pathway to prevent premature termination by tuning Pcf11 versus Nrd1 association with Pol II.

    Evidence Quantitative proteomics network analysis, ChIP-seq, RNA-seq, and rrp6Δ epistasis in yeast

    PMID:32187185

    Open questions at the time
    • Whether termination role requires catalysis untested
    • Human termination relevance unknown
  10. 2021 High

    Provided the high-resolution mammalian structural basis for RPAP2 action, locating it at the RPB1/RPB5 jaw interface in a position displaced upon PIC formation.

    Evidence 2.8 Å cryo-EM of mammalian Pol II–human RPAP2 complex

    PMID:34021257

    Open questions at the time
    • Catalytic engagement of CTD not captured
    • Functional consequence of displacement only inferred
  11. 2022 High

    Revealed a catalysis-independent function: RPAP2 sterically blocks TFIIF and inhibits PIC assembly, with depletion causing promoter Pol II/TFIIF accumulation.

    Evidence Cryo-EM, in vitro transcription, biochemical binding, and RPAP2-depletion ChIP-seq

    PMID:35476980

    Open questions at the time
    • Coordination between gatekeeping and phosphatase roles unclear
    • How RPAP2 is removed at the right time unknown
  12. 2022 Medium

    Expanded RPAP2/Rtr1 function to Pol II biogenesis, showing phosphatase-independent roles in Rpb1-Rpb2 and Rpb4/7 assembly that link to nuclear import and mRNA decay.

    Evidence Genetic suppressor analysis, Co-IP, microscopy, catalytic-dead mutants, and mRNA stability assays in yeast

    PMID:35216121 PMID:36190433

    Open questions at the time
    • Mechanistic role with Gpn3/Npa3 not structurally defined
    • Conservation of assembly role in humans untested
  13. 2022 Medium

    Identified a transcription-independent role as a PERK-dependent IRE1α phosphatase in the unfolded protein response, linking RPAP2 to stress signaling.

    Evidence Pharmacological and genetic manipulation with phosphorylation readouts in keratinocytes and mouse model

    PMID:35975910

    Open questions at the time
    • Direct biochemical IRE1α dephosphorylation by RPAP2 not demonstrated
    • Relationship to Pol II role unknown
    • Single lab
  14. 2025 High

    Defined how RPAP2 abundance is controlled, establishing an FBXW7/USP7 ubiquitin axis gated by p38/GSK3 phosphorylation and showing RPAP2 is the causal effector of cystogenesis upon FBXW7 loss.

    Evidence Ubiquitylation assays, phosphosite mutagenesis, pharmacology, and conditional double-knockout mouse epistasis

    PMID:39932049

    Open questions at the time
    • Which RPAP2 activity drives cystogenesis not pinpointed
    • Tissue specificity of the axis unexplained

Open questions

Synthesis pass · forward-looking unresolved questions
  • Whether RPAP2/Rtr1 acts through intrinsic catalytic phosphatase activity or principally as a non-catalytic cofactor/gatekeeper remains directly unresolved across its transcriptional and signaling roles.
  • Conflicting structural/biochemical activity data not reconciled
  • Catalysis-dependent versus -independent roles not cleanly separated
  • Mechanistic basis of putative IRE1α phosphatase activity undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 4 GO:0016787 hydrolase activity 3 GO:0098772 molecular function regulator activity 2
Localization
GO:0005634 nucleus 2 GO:0005829 cytosol 2
Pathway
R-HSA-74160 Gene expression (Transcription) 3 R-HSA-8953854 Metabolism of RNA 3 R-HSA-392499 Metabolism of proteins 1
Partners
FBXW7GPN3INTEGRATORNPA3RPB1RPB5RPB6USP7

Evidence

Reading pass · 17 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2008 Rtr1 (yeast RPAP2 ortholog) physically associates with active RNAPII transcriptional complex, shuttles between cytoplasm and nucleus, and is required for inducible transcription (GAL1 promoter). High-copy suppressors of rtr1Δ temperature-sensitive phenotype included core RNAPII subunits RPB5, RPB7, and RPB9, placing Rtr1 functionally within the RNAPII complex. Co-immunoprecipitation, genetic suppressor screen, growth assays, fluorescence microscopy Eukaryotic cell Medium 18408053
2009 Rtr1 (yeast RPAP2 ortholog) functions as a CTD phosphatase that dephosphorylates Ser5-phosphorylated RNAPII, localizes within coding regions between the peaks of Ser5-P and Ser2-P, and is essential for the transition from Ser5 to Ser2 CTD phosphorylation during transcription elongation. Deletion of Rtr1 causes accumulation of Ser5-P RNAPII, decreased transcription, and termination defects. ChIP, whole-cell extract phosphorylation analysis, deletion mutant phenotypic analysis, functional characterization of phosphatase activity Molecular cell High 19394294
2011 Human RPAP2 specifically recognizes phospho-Ser7 on the Pol II CTD and is recruited to snRNA genes via this mark. RPAP2 also interacts with Integrator complex subunits and functions as a CTD Ser5 phosphatase. siRNA knockdown of RPAP2 and Ser7-to-Ala mutation cause similar defects in snRNA gene expression, indicating Ser7-P recruits RPAP2, which in turn recruits Integrator and dephosphorylates Ser5. siRNA knockdown, ChIP, co-immunoprecipitation, in vitro phosphatase assay, mutational analysis Molecular cell High 22137580
2012 Crystal structure of Kluyveromyces lactis Rtr1 reveals a new type of zinc finger protein with no identifiable active site and no close structural homologues. Extensive in vitro experiments failed to detect CTD phosphatase activity, suggesting Rtr1 may have a non-catalytic role in CTD dephosphorylation. X-ray crystallography, in vitro phosphatase assays Nature communications High 22781759
2014 Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. In vitro, Rtr1 dephosphorylates both Ser5 and Tyr1 on the CTD (dual specificity). A single amino acid mutation reducing activity causes the same in vivo phenotype as full gene deletion, establishing that enzymatic activity is functionally important. In vitro phosphatase assay, site-directed mutagenesis, yeast complementation assay Journal of molecular biology High 24951832
2014 Rtr1 preferentially interacts with hyperphosphorylated RNAPII as its primary binding partner in yeast. Interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains lacking CTD Ser2 kinase, suggesting Ser2 CTD phosphorylation is required for Rtr1 recruitment during transcription elongation. Quantitative proteomics (mass spectrometry-based interactome), affinity purification Molecular bioSystems Medium 24671508
2014 The C-terminal region of human RPAP2 interacts directly with Pol II subunit Rpb6. RPAP2 occupies coding and 3' regions of protein-coding genes (MYC, GAPDH), and siRNA-mediated knockdown of RPAP2 causes defects in pre-mRNA 3'-end formation. Direct interaction assay (pulldown), ChIP, siRNA knockdown, RNA processing analysis Drug discoveries & therapeutics Medium 25639305
2016 Crystal structure of S. cerevisiae Rtr1 at 2.6 Å resolution reveals a phosphoryl transfer domain with a putative active site containing a trapped sulfate ion in a deep groove between the zinc finger domain and a pair of helices. Mutagenesis of active-site residues disrupts in vitro catalytic activity and fails to rescue growth of rtr1Δ yeast. RPAP2 and a mutant of the conserved catalytic site show similar behavior, indicating a conserved reaction mechanism distinct from other phosphatase families. X-ray crystallography (2.6 Å), site-directed mutagenesis, in vitro phosphatase assay, yeast complementation Science signaling High 26933063
2016 Rtr1 is a global regulator of CTD Ser5 phosphorylation; RTR1 deletion causes genome-wide increases in Ser5-P and global increases in cotranscriptional H3K36 trimethylation, consistent with Rtr1 controlling the number of binding sites for histone methyltransferase Set2. ChIP-chip (chromatin immunoprecipitation with microarrays), genome-wide analysis of RNAPII phosphorylation Molecular and cellular biology Medium 27247267
2020 Loss of RTR1 alters interactions within the termination machinery: Rtr1 deletion decreases RNAPII-Pcf11 (CF1A subunit) interactions and increases RNAPII-Nrd1 interactions. RTR1 deletion globally increases termination at noncoding genes via the NNS (Nrd1-Nab3-Sen1) pathway and causes premature termination at protein-coding genes. Rtr1 normally restricts NNS-dependent termination to prevent premature termination. DisCo network analysis (quantitative proteomics), genome-wide ChIP-seq (RNAPII and Nrd1 occupancy), RNA-seq, genetic epistasis with rrp6Δ PLoS genetics High 32187185
2021 Cryo-EM structure of mammalian Pol II in complex with human RPAP2 at 2.8 Å resolution shows RPAP2 binds between the jaw domains of RPB1 and RPB5 subunits. RPAP2 is incompatible with downstream DNA binding during transcription and is displaced upon pre-initiation complex formation. Cryo-electron microscopy (2.8 Å resolution) Communications biology High 34021257
2022 RPAP2 binds both hypo- and hyper-phosphorylated Pol II. Cryo-EM structure of the RPAP2-Pol II complex shows mutually exclusive assembly with the pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents and disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation. Loss of RPAP2 causes global accumulation of TFIIF and Pol II at promoters, demonstrating RPAP2 inhibits PIC assembly independent of phosphatase activity. Cryo-EM structure, in vitro transcription assay, RPAP2 depletion with ChIP-seq, biochemical binding assays Cell reports High 35476980
2022 Rtr1 is required for assembly of the two largest RNAPII subunits (Rpb1-Rpb2) by cooperating with assembly factors Gpn3 and Npa3. RTR1 overexpression is a multicopy suppressor of gpn3, gpn2, and rba50 assembly mutants. Deletion of RTR1 leads to cytoplasmic clumping of RNAPII subunits. Notably, phosphatase-dead Rtr1 mutant does not trigger cytoplasmic clumping, indicating this assembly function is independent of phosphatase activity. Genetic suppressor analysis, co-immunoprecipitation, fluorescence microscopy, catalytically inactive mutant analysis FASEB journal Medium 36190433
2022 Rtr1 mediates the association of the Rpb4/7 heterodimer with the rest of RNAPII. RTR1 deletion alters RNAPII assembly, increasing chromatin-associated RNAPII lacking Rpb4, which decreases Rpb4-mRNA imprinting and consequently increases mRNA stability. This establishes a link between Rtr1-mediated RNAPII assembly and mRNA decay regulation. Co-immunoprecipitation, ChIP, mRNA stability assays, genetic deletion analysis International journal of molecular sciences Medium 35216121
2022 RPAP2 functions as a PERK-dependent IRE1α phosphatase in the unfolded protein response. TGFβ1-mediated dephosphorylation of IRE1α is mediated through PERK via RPAP2, as shown by pharmacological and genetic approaches in keratinocytes expressing oncogenic HRas. Pharmacological inhibition, genetic knockdown/overexpression, phosphorylation assays in cell culture and in vivo mouse model Molecular carcinogenesis Medium 35975910
2025 FBXW7 E3 ligase targets RPAP2 for polyubiquitylation and proteasomal degradation after RPAP2 is pre-phosphorylated at Ser562 by p38 and at Thr565 by GSK3. USP7 deubiquitylates and stabilizes RPAP2. HSP90 inhibition promotes RPAP2 degradation by CRL5-FBXW7. Hepatic-specific deletion of Fbxw7 causes cystogenesis with RPAP2 accumulation, and simultaneous Rpap2 deletion reverses cystogenesis, establishing RPAP2 as the causal downstream effector. Co-immunoprecipitation, ubiquitylation assays, site-directed mutagenesis (Ser562/Thr565), pharmacological inhibition, conditional knockout mouse model (genetic epistasis) Advanced science High 39932049
2025 RPAP2 functions as a transcription-specific cofactor for influenza A virus polymerase, distinct from replication-specific cofactors, as identified by differential interactome screening. Differential interactome screen (comparative proteomics), functional characterization bioRxivpreprint Low bio_10.1101_2025.06.06.658254

Source papers

Stage 0 corpus · 18 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 Rtr1 is a CTD phosphatase that regulates RNA polymerase II during the transition from serine 5 to serine 2 phosphorylation. Molecular cell 121 19394294
2011 Ser7 phosphorylation of the CTD recruits the RPAP2 Ser5 phosphatase to snRNA genes. Molecular cell 110 22137580
2012 The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity. Nature communications 40 22781759
2014 Rtr1 is a dual specificity phosphatase that dephosphorylates Tyr1 and Ser5 on the RNA polymerase II CTD. Journal of molecular biology 38 24951832
2008 Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins. Eukaryotic cell 34 18408053
2014 The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae. Molecular bioSystems 31 24671508
2022 RPAP2 regulates a transcription initiation checkpoint by inhibiting assembly of pre-initiation complex. Cell reports 19 35476980
2014 Human RNA polymerase II-associated protein 2 (RPAP2) interacts directly with the RNA polymerase II subunit Rpb6 and participates in pre-mRNA 3'-end formation. Drug discoveries & therapeutics 17 25639305
2021 Cryo-EM structure of mammalian RNA polymerase II in complex with human RPAP2. Communications biology 15 34021257
2020 RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination. PLoS genetics 15 32187185
2016 Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase. Science signaling 15 26933063
2016 Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation. Molecular and cellular biology 14 27247267
2025 The FBXW7-RPAP2 Axis Controls the Growth of Hepatocellular Carcinoma Cells and Determines the Fate of Liver Cell Differentiation. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 7 39932049
2022 Rtr1 is required for Rpb1-Rpb2 assembly of RNAPII and prevents their cytoplasmic clump formation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 6 36190433
2022 The Association of Rpb4 with RNA Polymerase II Depends on CTD Ser5P Phosphatase Rtr1 and Influences mRNA Decay in Saccharomyces cerevisiae. International journal of molecular sciences 5 35216121
2022 TGFβ1 regulates HRas-mediated activation of IRE1α through the PERK-RPAP2 axis in keratinocytes. Molecular carcinogenesis 5 35975910
2020 Active Monomer RTR-1 Derived from the Root of Rhodomyrtus t omentosa Induces Apoptosis in Gastric Carcinoma Cells by Inducing ER Stress and Inhibiting the STAT3 Signaling Pathway. Cancer management and research 4 32440210
2025 Rtr1 and RPAP2: versatile players in transcription and more. Transcription 0 41313435

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