Multi-step coordination of telomerase recruitment in fission yeast through two coupled telomere-telomerase interfaces

Thank you for submitting your article "Telomerase Recruitment is Promoted by a Bivalent Transition-State Involving both Ccq1 and the TEL-Patch of Tpz1" for consideration by eLife. Your article has been favorably evaluated by James Manley (Senior editor) and three reviewers, one of whom, Kathleen Collins, is a member of our Board of Reviewing Editors. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

(Note that the Title should be changed as part of the clarifications of essential revision point number 6.)

Summary:

In this thorough and thoughtful study, the authors illuminate interaction trade-offs that unify previously puzzling requirements for telomerase action at telomeres in fission yeast. Using elegant genetic and biochemical approaches, they demonstrate a series of interactions and interaction rearrangements between telomere and telomerase proteins that load and then activate telomerase for end-elongation. Biochemical specificity of interactions accounts for how short telomeres are preferentially elongated in a cell cycle dependent manner. Results from fission yeast studied here provide ideas about telomerase-telomere interaction across species.

Essential revisions:

1) The Tpz1-Ccq1-Est1 ternary complex should be described as speculative in existence or it should be demonstrated directly. Is it possible to directly detect the formation of the ternary complex Est1-Tpz1-Ccq1 using a pull-down of Ccq1 to show that Est1 and Tpz1-C can be pulled down in a Ccq1-dependent manner? Alternatively, since the authors are already using radio-labeled Est1p, perhaps this reagent could be used as the basis for some form of super-shift EMSA experiment to demonstrate formation of the ternary complex?

2) Figure 1D should better document the senescent phenotype that is reported. The authors should analyze the telomere length in the tpz1-R81E/poz1D strain at later generations to make certain that the strain senesces or show that the telomeres are dependent on homologous recombination. In addition, "phenocopying" may not be the correct word to describe similar phenotypes resulting from different mutations.

3) The quantification shown in Figure 2C and D should normalize Trt1 and Ccq1 in the IP to Tpz1 bound to beads to compensate for mutant effects on protein stability and IP efficiency before making comparisons between strains.

4) Figure 5C experiment should be re-run in a way that normalization with Input levels can be performed, or the statement that Ccq1-T93A and Est1-K252E do not interact should be qualified to reflect the technical limitation. Est1-K252E seems significantly reduced compared to wild type and other Est1 mutants in the ccq1-T93A background.

5) In their concluding model (Figure 7) the authors speculate that Ccq1 and Est1 leave the telomere while Tpz1 stays telomere bound. This data contradicts the study's cell cycle Co-IP data (Figure 6B) that shows Tpz1 and Ccq1 are bound together throughout the cell cycle and the Est1-Ccq1 interaction data showing decreasing interaction during late S-phase (Figure 6E). In addition, data from the Nakamura lab shows Tpz1- and Ccq1-telomere interaction as co-incident with both protein telomere interaction peaking in late S-phase (Chang et al., 2013), which contradicts this model in which Ccq1 leaves the telomere while Tpz1 remains bound. These discrepancies with the model should be accounted for. Is only a minor fraction of the Tpz1-Ccq1 complex interacting with Est1 at any given time?

6) With respect to the nomenclature used to describe the proposed binding intermediate complex formed between Tpz1-Ccq1-Est1 (telomerase), the reviewers should strongly consider changing the name of the model. The term transition state has a very specific meaning, and is precisely not to be considered a detectable intermediate during a given reaction pathway. In contrast the complex being proposed in the present study, while possibly transient, would be considered a bone fide intermediate complex during telomerase recruitment. In addition, since the paper describes two distinct bivalent interactions, one within the Est1-Ccq1 complex and the second within the putative ternary complex of Tpz1-Ccq1-Est1(telomerase), it is potentially confusing to the reader what exactly is meant by 'bivalent transition state'. The authors might consider revising their wording to something more typical of a transiently formed multi-protein complex (i.e. telomerase pre-priming complex, telomerase recruitment complex, etc.).

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Telomerase Recruitment is Promoted by an Intermediate-State Involving both Ccq1 and the TEL-Patch of Tpz1" for further consideration at eLife. Your revised article has been favorably evaluated by James Manley (Senior editor) and a Reviewing editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Title:

The title "Telomerase Recruitment is Promoted by an Intermediate-State Involving both Ccq1 and the TEL-Patch of Tpz1" does not sufficiently communicate the import or advance of this work to a general audience. Something like "Multi-step coordination of telomerase recruitment and activation at fission yeast telomeres" extended by something like "through two conformationally coupled interaction interfaces."

Abstract:

Needs to be overhauled to communicate the actual content and importance of this work.

1) "[…]how the TEL-patch communicates with other shelterin components and telomerase holoenzyme to achieve temporally and spatially regulated telomerase recruitment is unclear"

This work is not about how the TEL-patch communicates with other shelterin components or how the TEL-patch communicates to telomerase, which is previously established. This work is about how TEL-patch interaction with telomerase integrates into the overall orchestration of telomerase regulation at chromosome ends.

2) Need to introduce what Ccq1 is, since it is not a household protein name. Could include the adjective telomere-associated or telomere-interacting before first use of Ccq1 in the Abstract.

3) "we discovered that telomerase recruitment is promoted by an intermediate-state.[…]" But recruitment is not "promoted" by an intermediate state – intermediate to what at this point also not explained – instead it is true that 'telomerase action at telomeres requires formation and resolution of an intermediate state.' "[…]to achieve timely and legitimate telomerase engagement" But what is legitimate? No illegitimate engagement is studied here – needs to be something like 'to achieve regulated and efficient telomerase elongation of telomeres.'

Revise "bivalent" and "transition-state"

Definitions of bivalent online: "having two combining sites" "a molecule formed from two or more atoms bound together as a single unitmolecule"

As Reviewers explained previously, the use of "bivalent" is confusing. The revision has not make "bivalent" more clear. It does not make sense to readers even within the telomere field to write "telomerase recruitment is promoted by a bivalent intermediate-state, formed via both Ccq1-Est1 and Tpz1 (TEL-patch)-Trt1 interactions." It would be sufficient and less confusion-inducing to say "is promoted by an intermediate-state with both Ccq1-Est1 and Tpz1 (TEL-patch)-Trt1 interactions."

The Discussion still begins with "A bivalent transition-state model for telomerase recruitment" despite Reviewers' indication to remove "transition-state" and refrain from vague uses of "bivalent."

Hyperbole and coherence

Last paragraph of Intro:

"Thus, the temporal and spatial information for telomerase recruitment is endowed to the TEL-patch through the phosphorylation status of Ccq1 Thr93, thus achieving cell cycle-specific and telomere length-dependent telomere elongation." This work does not direct address "spatial information" or "telomere length-dependent telomere elongation" – here and elsewhere, less vague/out-of-place summary statements would be more useful to understand what this work shows directly. In the Discussion is it appropriate to speculate about how telomere length would affect the two-pronged cooperative Ccq1 and Tpz1 recruitment of telomerase by changing the efficiency of Ccq1 phosphorylation – but there are no experiments that physically dissect length-dependent versus length-independent recruitment in this work.

First paragraph of Results, echoed in last sentence of Results:

"additional interactions between shelterin components and Trt1 must exist to engage Trt1 directly at the very 3' end of the telomere." "this in turn enables Tpz1 to directly position telomerase to the very 3' end of the telomere"

The discussion says that ssDNA might be required to engage Trt1 at the very end of a telomere – that is valid speculation as Discussion section content. But otherwise this work does not address the "very 3' end of the telomere" and therefore does not show a role for shelterin-Trt1 interaction in positioning telomerase at the very 3' end of a telomere. The quoted conclusions, and their like, need to be rephrased or deleted; they lack logic or fulfilment.

Discussion:

Starting from around "Tpz1 may also activate the telomerase-nonextendible state of telomeres through its interaction with Ccq1."

This section needs to be clarified, because return of the telomere to non-extendible state is not described anywhere else and so the ideas need to be spelled out more clearly.

"Moreover, telomerase activation function was also demonstrated for the Tpz1 OB-fold domain (Armstrong et al., 2014), which is close to the TEL-patch."

But the TEL patch is within the OB fold domain. Please spell out the point about activation.

Next paragraph, this sentence is out of place and should be deleted "conservation of the TEL-patch in fission yeast Tpz1 (TPP1 in humans) further supports the similarity of telomerase recruitment pathways in the human and fission yeast systems." The rest of the paragraph is about things other than the TEL patch.

Overall:

This is not an eLife paper until it is revised with sentence-by-sentence reflection on the verity of logic and on the accuracy of presentation/typos.

E.g. of just one example of the former, flip first two lines of Introduction for accuracy. "Telomeres are essential[…] In most eukaryotes, telomeres are comprised of short tandem.[…]"

E.g. of just one example of the latter, Dyskeratosis congenital is not a disease.

Also fix the lack of abbreviation definitions (e.g. dsDNA, ssDNA) and noun/verb disagreement or mixed verb tenses.

Would be useful to split the long run-on paragraphs, such as the final paragraph of the Introduction and the first paragraph of the subsection “Est1 and Ccq1 form a stable complex through two binding sites”.