TAP65 (p65) is the La-related protein 7 (LARP7) family subunit of the Tetrahymena thermophila telomerase holoenzyme. It is a 542-residue protein built from three RNA-binding modules - an HTH La-type domain, a classical RRM, and a C-terminal xRRM that this protein defined structurally - which act together to recognise telomerase RNA (TER). p65 is a non-catalytic subunit: together with the reverse transcriptase TERT and TER it forms the three-component catalytic core of the holoenzyme, which in the intact enzyme is tethered through p50 to an RPA-related Teb1-Teb2-Teb3 heterotrimer and a CST-like p75-p45-p19 heterotrimer. Its activity is RNA chaperoning rather than catalysis. The La-type and RRM modules clamp the 3' oligo(U) terminus of the Pol III-transcribed TER precursor and shield it from exonucleolytic degradation, which is what sets the cellular level of telomerase RNA; the xRRM then reads the conserved GA bulge of proximal stem IV and bends the RNA by about 105 degrees. That bend converts TER from a conformationally heterogeneous molecule into the single fold that TERT can engage, so the enzyme assembles in a fixed order - p65 onto TER first, then TERT - with roughly thirty-fold higher TERT affinity than for naked TER. p65 remains in the assembled particle, holding TER and TERT in their active conformations. Loss of p65 is lethal in Tetrahymena: telomerase RNA fails to accumulate, TERT is destabilised in its absence, and telomeres become critically short. The protein is specific to telomerase RNA and does not behave as a general La protein acting on RNA polymerase III transcripts.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000781 chromosome, telomeric region | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Telomeric-chromatin localization inferred by UniProt from its subcellular-location vocabulary; plausible for a constitutive holoenzyme subunit, but peripheral to what p65 is shown to do. Reason: This row comes from the UniProtKB-SubCell SL-0276 mapping, which rests on UniProt's own ECO:0000305 inference rather than an observation of p65 on telomeric chromatin. The inference is reasonable: p65 is a stable, RNase-resistant subunit of the holoenzyme (PMID:19941821) whose depletion shortens telomeres (PMID:15131081), so the particle it belongs to does act at chromosome ends. It is retained at this level of precision because nothing finer is warranted. It is marked non-core because every assayed p65 activity - 3'-end protection of TER, TER folding, and hierarchical TERT loading - belongs to ribonucleoprotein biogenesis, upstream of telomere engagement, and no direct localization of Tetrahymena p65 to the telomeric region has been published. Supporting Evidence: PMID:19941821 RNase treatment of p65fzz holoenzyme depleted all holoenzyme subunits except p65 PMID:15131081 We found that telomeres were dramatically shorter in p65 and p45 knockdown strains |
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | MODIFY | Summary: True but far less specific than the demonstrated activity: p65 binds telomerase RNA, and essentially only telomerase RNA. Reason: The InterPro IPR035979 (RBD domain superfamily) mapping is correct in essence but is pitched at the level of the fold rather than the evidence. The specific supported descendant, GO:0070034 telomerase RNA binding, is already carried by this gene six times over by IDA, and QuickGO confirms GO:0003676 is an ancestor of GO:0070034, so the replacement is a strict refinement rather than a lateral move. The specificity is not an assumption: affinity-purified p65 recovers TER essentially to the exclusion of other RNAs, knockdown leaves other RNA polymerase III transcripts untouched, and a size-matched structured control RNA fails to compete in vitro. Proposed replacements: telomerase RNA binding Supporting Evidence: PMID:15131081 The similarity of the TERT- and p65-associated RNA profiles suggests that both proteins are preferentially associated with telomerase RNA. PMID:15131081 whereas telomerase RNA level decreased in p65 knockdown strains, the accumulation of other RNA polymerase III transcripts was unaffected PMID:15696174 we determined that Escherichia coli 5S RNA was incapable of p65 mobility shift competition |
| GO:0003723 RNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Family-level RNA binding is correct, but the substrate is known: p65 is a telomerase-RNA-specific binder, not a general La-type Pol III transcript binder. Reason: The RRM (IPR000504), xRRM (IPR014886) and PANTHER PTN008582015 evidence establishes RNA binding at the family level, which is true. For this protein the substrate has been determined, so the informative term is the descendant GO:0070034 telomerase RNA binding, which the gene already carries by IDA. This refinement matters for a LARP7-family protein in particular: the obvious family-based reading would make p65 a ciliate La binding Pol III transcripts generally, and that reading was tested and rejected in Tetrahymena - TER levels collapse on p65 knockdown while 5S rRNA, SRP RNA, U2, U3 and U6 are unaffected. Binding is high-affinity and sequence-specific for proximal stem IV, with four separable modules of the protein contributing. Proposed replacements: telomerase RNA binding Supporting Evidence: PMID:15131081 whereas telomerase RNA level decreased in p65 knockdown strains, the accumulation of other RNA polymerase III transcripts was unaffected PMID:15696174 These results demonstrate that p65 recognizes the sequence of proximal stem IV. PMID:16507983 We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:15131081 Holoenzyme proteins required for the physiological assembly ... | ACCEPT | Summary: p65 was identified in affinity-purified endogenous Tetrahymena telomerase and shown by reciprocal immunodepletion to be a stable, not transient, component of the particle. Reason: This is the founding identification of p65 as a holoenzyme subunit. Affinity purification of epitope-tagged, endogenously assembled telomerase recovered it, and serial immunodepletion with anti-p65 removed the bulk of telomerase activity and telomerase RNA from extract, establishing that the predominant physiological pool of the RNP contains p65 rather than that p65 is a purification artifact. Supporting Evidence: PMID:15131081 This finding demonstrates that both proteins are stable telomerase RNP components PMID:15131081 We find that p65 in cell extract is associated specifically with telomerase RNA, and that genetic depletion of p65 reduces telomerase RNA accumulation in vivo. |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:15696174 A telomerase holoenzyme protein enhances telomerase RNA asse... | ACCEPT | Summary: Reconstitution with recombinant subunits shows p65 is part of a TERT-TER-p65 ternary particle, the catalytic core of the holoenzyme. Reason: The paper assembles the complex from defined components and shows that tagged p65 copurifies untagged TERT and vice versa, strictly dependent on a bridging TER molecule. Membership in the complex is therefore demonstrated directly rather than inferred from co-fractionation. Supporting Evidence: PMID:15696174 These findings demonstrate that in the presence of TER, p65 and TERT associate to form a ternary complex. |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:16507983 A novel RNA binding domain in tetrahymena telomerase p65 ini... | ACCEPT | Summary: Quantitative assembly assays place p65 in the TER-containing holoenzyme core and define the order in which it joins. Reason: The study dissects p65 modules by their contribution to TER affinity and to TERT recruitment, and shows p65 and TERT occupy the same TER molecule in a fixed order. This is direct evidence for p65 as a constituent of the assembled particle. Supporting Evidence: PMID:16507983 we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding PMID:16507983 We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:17322903 Stepwise protein-mediated RNA folding directs assembly of te... | ACCEPT | Summary: Single-molecule observation of complex formation shows p65 within the functional TER-p65-TERT ternary complex. Reason: The cached record for this reference is abstract-only (full_text_available: false), so the full experimental detail the curator read is not available here. The abstract nonetheless states directly that p65 participates in formation of the functional ternary complex, and the claim is independently established by PMID:15696174 and the later cryo-EM structures. There is no basis for doubting the curator's IDA. Supporting Evidence: PMID:17322903 interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex PMID:15696174 These findings demonstrate that in the presence of TER, p65 and TERT associate to form a ternary complex. |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:19941821 An RPA-related sequence-specific DNA-binding subunit of telo... | ACCEPT | Summary: Holoenzyme composition mapping with tagged subunits places p65 in the RNP catalytic core, which in turn docks onto the p75-p45-p19 subcomplex. Reason: Tagging p65 itself and treating the purified holoenzyme with RNase showed that every other subunit is lost while p65 remains, identifying p65 as the subunit whose association survives independently of the RNA scaffold and confirming its place in the particle. Supporting Evidence: PMID:19941821 RNase treatment of p65fzz holoenzyme depleted all holoenzyme subunits except p65 PMID:19941821 a biologically stable assembly of the RNP catalytic core (p65-TER-TERT) associates directly with a second holoenzyme protein subcomplex (p75-p45-p19) |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:20713447 Tetrahymena telomerase protein p65 induces conformational ch... | ACCEPT | Summary: Biochemical reconstitution with purified p65, TERT and TER variants shows p65 within the assembled, catalytically competent RNP. Reason: This cached record is abstract-only (full_text_available: false). The abstract establishes that p65 is a constituent of the assembled RNP and not merely a transient assembly factor, describing it as remaining in the particle and stabilizing TER and TERT together. Complex membership is corroborated by the purification and structural studies. Supporting Evidence: PMID:20713447 p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:23552895 The architecture of Tetrahymena telomerase holoenzyme. | ACCEPT | Summary: Negative-stain EM reconstruction of the intact holoenzyme resolves p65 as a positioned subunit of the particle. Reason: Subunit-specific Fab labelling assigned density to p65 within the whole holoenzyme and the reconstruction partitions the enzyme into functional units with p65 in the catalytic core. This is as direct as complex-membership evidence gets. Supporting Evidence: PMID:23552895 The EM results together with reconstituted enzyme activity assays suggest a subdivision of holoenzyme functional units: the TERT-TER-p65 catalytic core, p50-Teb1, and 7-1-4. |
| GO:0005697 telomerase holoenzyme complex | IDA PMID:26472759 Structure of Tetrahymena telomerase reveals previously unkno... | ACCEPT | Summary: Cryo-EM structure of Tetrahymena telomerase with p65 built into the TERT-TER-p65 catalytic core. Reason: The structure enumerates the holoenzyme subunits and builds a pseudoatomic model of the catalytic core containing p65 bound to stem-loop 4 of TER. Membership is established at near-atomic resolution. Supporting Evidence: PMID:26472759 In addition to TERT and TER, the Tetrahymena telomerase holoenzyme contains six other known proteins: p65, p75, p45, p19, p50, and Teb1 PMID:26472759 inducing a large bend in the RNA that facilitates assembly of TERT with TER to form the RNP catalytic core |
| GO:0007004 telomere maintenance via telomerase | IDA PMID:15696174 A telomerase holoenzyme protein enhances telomerase RNA asse... | ACCEPT | Summary: p65 acts in the telomerase-mediated telomere maintenance pathway by building the enzyme that performs it; this row supplies the mechanism behind the in vivo phenotype. Reason: Taken alone, an in vitro reconstitution does not observe telomeres, so this row is weaker on its own than the IMP row for the same term. It is accepted rather than downgraded for two reasons. First, the two rows assert the same claim and must be treated consistently. Second, this paper supplies exactly what the IMP leaves open: it shows p65 is what makes TERT load onto TER efficiently, roughly thirty-fold, so the short-telomere phenotype of p65 depletion is explained by a direct contribution of p65 to producing functional telomerase rather than by an indirect effect. p65 is an executing participant in this pathway, not merely something the pathway requires. Supporting Evidence: PMID:15696174 Notably, the p65–TER complex recruits TERT much more efficiently than does TER alone. PMID:15696174 TERT(1–516) bound to the p65–TER complex with ~30-fold greater affinity and more homogeneity than it bound to TER alone |
| GO:0007004 telomere maintenance via telomerase | IMP PMID:15131081 Holoenzyme proteins required for the physiological assembly ... | ACCEPT | Summary: TAP65 is essential, and its knockdown causes the canonical telomerase-loss phenotype of critically short, poorly elongating telomeres. Reason: This is the primary physiological evidence for the gene. Gene disruption could not be driven to completion, showing TAP65 is essential, and maximally assorted knockdown strains carry dramatically shortened telomeric repeat tracts with loss of the normal per-generation elongation. Because the lesion is specifically a failure to accumulate telomerase RNA, the phenotype places p65 inside the telomerase-mediated maintenance pathway rather than in a parallel end-protection process. Core. Supporting Evidence: PMID:15131081 The persistence of the wild-type loci through phenotypic assortment indicates that both p65 and p45 are encoded by essential genes PMID:15131081 We found that telomeres were dramatically shorter in p65 and p45 knockdown strains PMID:15131081 These findings provide genetic evidence that p65 functions in vivo to promote telomerase RNA accumulation |
| GO:0070034 telomerase RNA binding | IDA PMID:15131081 Holoenzyme proteins required for the physiological assembly ... | ACCEPT | Summary: Endogenous p65 associates specifically with telomerase RNA in cell extract. Reason: Two-step affinity purification of p65-TAP recovered a single predominant RNA species matching TER and matching the TERT-associated profile, with no recovery of the other abundant small RNAs. This is direct evidence of binding to the telomerase RNA and, importantly, evidence that the binding is selective rather than La-like promiscuity. Supporting Evidence: PMID:15131081 The similarity of the TERT- and p65-associated RNA profiles suggests that both proteins are preferentially associated with telomerase RNA. PMID:15131081 whereas telomerase RNA level decreased in p65 knockdown strains, the accumulation of other RNA polymerase III transcripts was unaffected |
| GO:0070034 telomerase RNA binding | IDA PMID:15696174 A telomerase holoenzyme protein enhances telomerase RNA asse... | ACCEPT | Summary: Recombinant p65 binds TER directly with low-nanomolar affinity and sequence specificity for proximal stem IV. Reason: Gel-shift assays with purified protein give an affinity of a few nanomolar at physiological ionic strength, competition assays localize the specificity to proximal stem IV, and an unrelated structured RNA of similar size does not compete. This is the quantitative basis for the term. Supporting Evidence: PMID:15696174 we determined that p65 bound TER with an affinity of ~2–5 nM under a range of conditions including physiological ionic strength PMID:15696174 These results demonstrate that p65 recognizes the sequence of proximal stem IV. PMID:15696174 we determined that Escherichia coli 5S RNA was incapable of p65 mobility shift competition |
| GO:0070034 telomerase RNA binding | IDA PMID:16507983 A novel RNA binding domain in tetrahymena telomerase p65 ini... | ACCEPT | Summary: Four separable modules of p65 contribute TER affinity, with the C-terminal domain reading the conserved central stem IV bulge. Reason: The quantitative dissection shows the binding is distributed across the protein rather than residing in a single motif, and identifies the specific RNA element each part recognizes. Binding to telomerase RNA is demonstrated directly throughout. Supporting Evidence: PMID:16507983 We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. PMID:16507983 This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER. |
| GO:0070034 telomerase RNA binding | IDA PMID:17322903 Stepwise protein-mediated RNA folding directs assembly of te... | ACCEPT | Summary: Single-molecule FRET shows p65 binding TER and remodelling it. Reason: The cached record is abstract-only (full_text_available: false), so the binding assays the curator read are not available here. The abstract states that p65 interacts with telomerase RNA and induces its structural rearrangement, which entails the binding, and the claim is redundantly established by the in vitro and structural work on the same interaction. Supporting Evidence: PMID:17322903 interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex PMID:15696174 we determined that p65 bound TER with an affinity of ~2–5 nM under a range of conditions including physiological ionic strength |
| GO:0070034 telomerase RNA binding | IDA PMID:20713447 Tetrahymena telomerase protein p65 induces conformational ch... | ACCEPT | Summary: Nuclease protection and mutational analysis with purified p65 map its contacts on TER to stems I and IV and beyond. Reason: The cached record is abstract-only (full_text_available: false). The abstract reports p65 binding to stems I and IV of telomerase RNA and interaction with regions of TER beyond stem IV, which directly supports the binding term independently of the assembly consequences. Supporting Evidence: PMID:20713447 the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER |
| GO:0070034 telomerase RNA binding | IDA PMID:22705372 Structural basis for telomerase RNA recognition and RNP asse... | ACCEPT | Summary: Crystal and NMR structures of the p65 C-terminal xRRM bound to TER stem IV give the interaction at atomic resolution. Reason: Structures of the protein-RNA complex, plus mutagenesis of Tyr407 and Arg465 that reduces stem IV binding, establish the molecular detail of telomerase RNA recognition by a combination of single-stranded and double-stranded contacts. A further La-type module reads the 3' oligo(U) tail. Supporting Evidence: PMID:22705372 RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER PMID:26472759 The p65 La-RRM1 binds the 3′ polyU tail |
| GO:0090669 telomerase RNA stabilization | IDA PMID:22705372 Structural basis for telomerase RNA recognition and RNP asse... | ACCEPT | Summary: p65 protects the 3' end of TER from degradation and is what sets the steady-state telomerase RNA level in vivo. Reason: The structural paper states the 3'-end protection role explicitly, and the La-type module is shown to engage the 3' oligo(U) tail, which is the physical basis for blocking exonuclease access. The in vivo counterpart is unambiguous: p65 knockdown collapses TER accumulation while leaving other small RNAs untouched, and TERT falls only secondarily as its RNA partner disappears. p65 performs this protection itself by occluding the RNA terminus, so this is participation and not merely a requirement. Core. Supporting Evidence: PMID:22705372 it protects the 3’ end of TER from degradation PMID:22705372 Tetrahymena p65 is a telomerase holoenzyme protein essential for TER accumulation in vivo PMID:15131081 telomerase RNA accumulation in the p65 knockdown strain was inhibited dramatically under selective pressure PMID:19941821 In vivo depletion of the La motif-containing p65, but not p45 or p75, reduced the biological accumulation of TERT and TER. |
| GO:1904868 telomerase catalytic core complex assembly | IDA PMID:15696174 A telomerase holoenzyme protein enhances telomerase RNA asse... | ACCEPT | Summary: p65 preassembles TER so that TERT loads onto it, raising TERT affinity for the RNA roughly thirty-fold. Reason: This is the defining demonstration of the assembly role. With entirely recombinant components, TERT binds the p65-TER complex about thirty-fold more tightly and more homogeneously than TER alone, and the effect requires the conserved central stem IV dinucleotide bulge, identifying the RNA element through which p65 acts. The term matches the claim exactly: what is assembled is the TERT-TER-p65 catalytic core. Supporting Evidence: PMID:15696174 TERT(1–516) bound to the p65–TER complex with ~30-fold greater affinity and more homogeneity than it bound to TER alone PMID:15696174 These assays reveal that p65 promotes TERT RNP assembly by a mechanism requiring the TER central stem IV dinucleotide bulge. PMID:15696174 Notably, the p65–TER complex recruits TERT much more efficiently than does TER alone. |
| GO:1904868 telomerase catalytic core complex assembly | IDA PMID:16507983 A novel RNA binding domain in tetrahymena telomerase p65 ini... | ACCEPT | Summary: Assembly is hierarchical, not cooperative: p65 binds TER first and the xRRM alone is necessary and sufficient to then recruit TERT. Reason: The paper separates p65's RNA-affinity modules from the module that drives TERT recruitment, and shows the C-terminal domain is both necessary and sufficient for the recruitment step. Establishing hierarchy rather than cooperativity is what makes p65 an agent of assembly rather than a passenger stabilized alongside TERT. Supporting Evidence: PMID:16507983 we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding PMID:16507983 This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER. |
| GO:1904868 telomerase catalytic core complex assembly | IDA PMID:17322903 Stepwise protein-mediated RNA folding directs assembly of te... | ACCEPT | Summary: Real-time single-molecule observation resolves the two sequential folding steps that order catalytic core assembly. Reason: The cached record is abstract-only (full_text_available: false). The abstract states the result directly: p65 binding induces a structural rearrangement of telomerase RNA which in turn directs TERT binding to form the functional ternary complex. This is the kinetic confirmation of the hierarchy reported biochemically in PMID:16507983. Supporting Evidence: PMID:17322903 interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex PMID:16507983 we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding |
| GO:1904868 telomerase catalytic core complex assembly | IDA PMID:20713447 Tetrahymena telomerase protein p65 induces conformational ch... | ACCEPT | Summary: p65 restricts TER to its active conformation and rescues assembly-defective TER and TERT mutants. Reason: The cached record is abstract-only (full_text_available: false). The abstract reports that p65 presents TERT with the active conformation of TER and that several TER mutants are active only when p65 is present, which is assembly-promoting activity demonstrated by rescue rather than merely by correlation. Supporting Evidence: PMID:20713447 by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER PMID:20713447 Several TER mutants exhibited telomerase activity only in the presence of p65 |
| GO:1904868 telomerase catalytic core complex assembly | IDA PMID:22705372 Structural basis for telomerase RNA recognition and RNP asse... | ACCEPT | Summary: The structure explains the mechanism: p65 bends TER stem IV by 105 degrees, positioning loop IV for TERT. Reason: The xRRM-stem IV structures show how a combination of single-stranded and double-stranded contacts wedges open the GA bulge and bends the RNA, and the paper lists bending stem-loop IV to position it for TERT as one of p65's essential roles in telomerase biogenesis. The cryo-EM holoenzyme structure shows the same geometry in the intact particle. Core. Supporting Evidence: PMID:22705372 RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER PMID:26472759 inducing a large bend in the RNA that facilitates assembly of TERT with TER to form the RNP catalytic core |
| GO:0140691 RNA folding chaperone | IDA PMID:22705372 Structural basis for telomerase RNA recognition and RNP asse... | NEW | Summary: p65 is an RNA folding chaperone: it binds TER and actively remodels it into the conformation competent for TERT loading, rescuing misfolded TER variants. Reason: GOA captures p65's binding (GO:0070034), the downstream assembly process (GO:1904868) and the stabilization process (GO:0090669), but no molecular function for the chaperone activity itself, which is the activity those processes depend on and the one the literature names in exactly these words. Participation test: p65 does the folding work itself rather than being folded or merely being required - its xRRM helix wedges the GA bulge open and bends the RNA by 105 degrees, and TER variants that cannot fold correctly on their own become active when p65 is supplied. Comparator test: QuickGO returns 36 experimental annotations to GO:0140691, including WRAP53/TCAB1 (UniProtKB:Q9BUR4, IDA) and mouse Wrap53 (UniProtKB:Q8VC51), telomerase-RNP biogenesis factors acting on telomerase RNA, as well as Hfq, Lsm2/3/4/8 and IMP3/IMP4; the term is therefore routinely applied to proteins in p65's role and its absence here is a gap rather than a convention. Non-redundancy: GO:0140691 sits under GO:0003674 and, via part_of, under the process GO:0034337 RNA folding; it is not a descendant of GO:0003723, so it is not entailed by the accepted GO:0070034, nor by the biological-process rows. Caveat, stated here so the row is self-contained: the discriminating chaperone test, whether substoichiometric p65 turns over multiple TER molecules, has not been done, and p65 stays in the assembled RNP as a molecular buttress rather than releasing after folding, which is atypical for a chaperone. The activity assay is solid; the catalytic-cycle question is open and is carried in suggested_experiments. The parent GO:0034337 is deliberately not also proposed. Supporting Evidence: PMID:22705372 it acts as a chaperone to correctly fold TER for protein binding PMID:22705372 RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER PMID:20713447 Several TER mutants exhibited telomerase activity only in the presence of p65 PMID:20713447 by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER PMID:17322903 interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex |
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Download this section (compressed HTML)Q: Should p65 also carry GO:0000333 telomerase catalytic core complex? The TERT-TER-p65 ternary core is a named, structurally resolved entity in every cryo-EM study, and GO:0000333 is more informative than GO:0005697. It was not proposed here because GO:0000333 is a part_of descendant of GO:0005697, which the gene already carries nine times by IDA, and CLAUDE.md directs against proposing a term that is a descendant of one the gene already holds. A GO-side convention on whether subcomplex and holoenzyme CC terms should coexist would settle it.
Suggested experts: Kathleen Collins, Juli Feigon
Q: Can the 3'-end protection and the stem IV bending functions of p65 be genetically separated? The question was posed explicitly when the assembly role was first described and has not been closed: are there p65 or TER variants that retain wild-type p65-TER and TERT-TER binding yet abolish p65 enhancement of TERT loading?
Suggested experts: Kathleen Collins, Michael D. Stone
Q: Does p65 remain associated with the holoenzyme at telomeres during repeat addition in vivo, or is its role confined to biogenesis? This determines whether GO:0000781 chromosome, telomeric region is a genuine site of action for p65 or only a property of the complex it builds.
Suggested experts: Kathleen Collins
Q: Does Tetrahymena encode a genuine La protein distinct from p65? A second La-motif locus was noted in the macronuclear genome but its expression and identity were never resolved, and the answer bears on how family-level RNA-binding inferences should be propagated within ciliates.
Suggested experts: Kathleen Collins
Experiment: Build p65 variants that uncouple the two activities - for example xRRM point mutants such as Y407A and R465A that lose stem IV binding while retaining the La-RRM1 clamp on the 3' oligo(U) tail, and reciprocal La-motif mutants that retain stem IV bending. Express each at endogenous levels in Tetrahymena and score TER accumulation by Northern blot, TER 3'-end integrity by 3' RACE, RNP assembly by TERT co-immunoprecipitation, and telomere length over continuous log-phase growth. A variant that preserves TER levels but fails TERT loading, or the converse, would establish the two roles as genetically distinct.
Hypothesis: The 3'-end protection and RNA-folding/TERT-loading activities of p65 are separable functions residing in different modules.
Type: Separation-of-function mutagenesis with in vivo complementation
Experiment: Use single-molecule FRET on TER pre-folded into misfolded states under conditions that trap them, then add p65 and measure whether it converts trapped molecules into the active conformation and whether it is released after folding. Pair this with a chaperone-standard assay: test whether substoichiometric p65 can catalytically turn over multiple TER molecules into the TERT-competent fold. Catalytic turnover would distinguish chaperone activity from stoichiometric conformational capture and would firm up the proposed GO:0140691 annotation.
Hypothesis: p65 acts as a true RNA folding chaperone, resolving kinetically trapped misfolded TER rather than only shifting a pre-existing conformational equilibrium.
Type: Single-molecule FRET refolding and substoichiometric turnover assay
Experiment: Tag endogenous p65 and TERT with orthogonal epitopes and perform chromatin immunoprecipitation against telomeric repeat DNA across the cell cycle, with TER-depleted and TERT-depleted strains as controls to separate RNA-dependent from protein-dependent recruitment. Complement with live-cell imaging of tagged p65 relative to a telomere marker. A p65 ChIP signal coincident with TERT would justify GO:0000781 as a site of action rather than an inference from complex membership.
Hypothesis: p65 is present at telomeres during repeat addition in vivo, not only during RNP biogenesis.
Type: Telomeric ChIP and live-cell colocalization
Experiment: Identify the second La-motif locus in the current macronuclear genome assembly, confirm it is transcribed by RT-PCR, tag the product and determine its bound RNA repertoire by CLIP or RIP-seq, asking specifically whether it binds the nascent 3' oligo(U) ends of 5S rRNA, SRP RNA and U6 while excluding TER. This would complete the division of labour that the original p65 paper left open.
Hypothesis: Tetrahymena encodes a genuine La protein, functionally distinct from p65, acting on RNA polymerase III transcripts generally.
Type: Gene identification plus RIP-seq/CLIP of the candidate La protein
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