TTF2 prevents premature rRNA synthesis during mitotic exit

  1. Catarina Pedro
  2. Laura Tovini
  3. Catarina Peneda
  4. Mia Maesano Krapinec
  5. Raquel A. Oliveira

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Abstract

Mitosis poses major challenges to cellular transcription. As cells enter mitosis, transcription is globally silenced and must be precisely restored upon mitotic exit. These processes are primarily regulated by Cdk1-dependent phosphorylation. In parallel, additional mechanisms, including Transcription Termination Factor 2 (TTF2)-mediated removal of nascent transcripts, reinforce transcriptional shutdown. How these layers of regulation control individual RNA polymerases and influence transcriptional reactivation at mitotic exit remains poorly understood.

Here, we probed how TTF2 differentially controls transcription of distinct RNA classes, using polymerase-specific perturbations and nascent RNA labelling across mitosis. Loss of TTF2 led to accumulation of chromatin-associated transcripts during metaphase, predominantly RNA Polymerase II–derived, consistent with its established role in transcriptional clearance. More unexpectedly, TTF2 depletion caused premature RNA Polymerase I reactivation during anaphase, resulting in unscheduled rRNA synthesis and early recruitment of nucleolar proteins. These findings place TTF2 as a novel regulator of RNA Polymerase I reactivation at mitotic exit. Disruption of this control persists beyond mitosis, resulting in increased nucleolar fragmentation in interphase. Together, these findings reveal TTF2 as a conserved regulator that interfaces with multiple RNA polymerases through functionally distinct modes of control, coordinating both transcriptional shutdown and timely reactivation across mitosis.

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  1. Review Commons

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, the authors investigate the role of Transcription Termination Factor 2 (TTF2) in the regulation of mitotic transcription. Using siRNA-mediated knockdown in two distinct human cell types combined with nascent RNA labeling (EU pulse), the authors identify an unexpected role for TTF2 in the timing of RNA Polymerase I (Pol I) reactivation following mitosis. The study suggests that this temporal misregulation may have downstream consequences for nucleolar morphology and function in interphase. The manuscript is well-written, and the figures are of high quality and clearly presented.

    Major comments:

    1. A primary limitation of the current study is that it does not deeply explore the underlying mechanism of the observed phenomenon. To strengthen the claims, the following points should be addressed:

    1a. Directionality of Phenotype: In Page 9, the authors conclude that TTF2 depletion is linked to abnormal nucleolar organization during interphase. It remains unclear if this is a direct result of mitotic misregulation or an independent interphase effect. To distinguish between these possibilities, I suggest the following experiment: perform a mitotic shake-off early in the siRNA treatment (~24h), collect mitotic cells, and allow them to re-enter G1 to image for nascent RNAs and nucleolin. This would clarify if the mitotic defect precedes and causes the interphase morphology changes. Alternatively, the authors should state that their current study cannot distinguish between these two possibilities.

    1b. Secondary Effects: The long duration of siRNA treatment (48h) raises the possibility that TTF2 knockdown misregulates the expression of other Pol I regulatory factors, leading to secondary effects. This limitation should be explicitly acknowledged in the Discussion.

    1. The term "significant" is used throughout the manuscript without accompanying statistical testing.

    2a. Please provide statistical analyses (e.g., p-values) for the average plots in Figures 1-3 to substantiate the findings.

    2b. Where statistics are not performed, the language should be softened to "notable" or "observed increase" rather than "significant."

    1. siRNA knockdowns are generally supported by quantification. Please provide the percentage reduction of the target protein by quantifying the blots provided in the supplemental figures.
    2. To rule out the possibility that the increased nucleolin signal observed after TTF2 KD is simply due to higher protein abundance, the authors should perform a western blot to confirm that total nucleolin protein levels remain unchanged upon TTF2 depletion.

    Minor comments:

    1. The abstract and discussion refer to the role of TTF2 as a "conserved" process. As the study only tested human cell lines, "conserved" is technically inaccurate (as it implies evolutionary comparison). I recommend using "general" or "cell-type independent."
    2. While the Methods section is detailed, the Results section would benefit from brief descriptions of the treatments to improve flow.

    Example revision (Page 4): "...we treated two distinct cell lines with control and TTF2-specific siRNAs for 48 hours, followed by a 30-minute EU pulse to label nascent RNAs. Click chemistry and Hoechst labeling enabled 2-color imaging of mitotic chromosomes and nascent RNA..."

    1. The data generally agree across both cell types; however, the presence of clustered signals in HeLa metaphase chromosomes is a notable divergence. It would be beneficial to include speculation in the Discussion on whether this represents a failure to silence Pol I transcription or an even earlier reactivation, and what this implies about a cancer cell line.

    Significance

    General assessment:

    The study is strong in its use of two different cell systems, providing confidence that the observed effects are not cell-line-specific. The figures are beautifully presented and the writing is clear. The primary limitation is the lack of mechanistic depth regarding how TTF2 specifically interfaces with the Pol I machinery compared to its known roles with Pol II.

    Advance:

    This work reports a previously unrecognized role for TTF2 in the temporal control of Pol I reactivation. While TTF2 is well-known for its role in terminating transcription and facilitating Pol II release during mitosis, its specific influence on the nucleolar transcription cycle provides a new perspective on how cells transition out of the mitotic state.

    Audience:

    This research will be of interest to researchers in the fields of gene regulation, the cell cycle, and nucleolar biology. Because it touches on the fundamental process of how transcriptional machinery is reset after cell division, it has implications for the broader basic research community interested in epigenetic memory and cellular identity.

  3. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this paper by the Oliveira lab a new perspective on TTF2 function during mitosis is proposed. Known for its role to terminate transcription at mitotic onset, this paper further shows an exciting involvement of TTF2 to schedule timely rDNA transcription at mitotic exit. Moreover, this role is shown to have a clear importance in the structuration of nucleoli, since TTF2 depletion is associated with premature partial assembly of nucleoli on the mitotic chromosomes and, subsequently, to fragmented nucleoli in interphase. These conclusions, which are well supported by imaging data, are original, interesting and, in fact, largely unexpected.

    The paper is very simple in its execution, based on siRNA depletion of TTF2 and monitoring of transcription by imaging using EU incorporation and rRNA-FISH, as well as nucleoli morphology and dynamics using immunostainings. Yet, it is well executed and has no major caveats. However, the authors should consider the following:

    1. The Teves lab has shown that TBP is a key factor maintaining its binding at rDNA loci during mitosis, enabling a prompt reactivation of rRNA production in interphase (Kwan et al. RNA 2024). This paper should be discussed on the light of current findings.
    2. In relation to the previous comment, I would strongly recommend the authors to analyse TBP-depleted cells, ideally using the line generated in the Teves lab, to address whether delayed rDNA transcription after mitosis leads to delayed nucleoli structuration. This assay would allow them to further confirm their model.
    3. In addition, it would be important to test if in the absence of mitotic TBP, the depletion of TTF2 does also lead to mitotic transcription.

    Significance

    Strengths: originality of the observation and simplicity of the experimental setup

    Limitations: exclusively based on imaging data

    Advance: completely unanticipated observation

    Audience: general readers interested in gene regulation

    My expertise: gene regulation through mitosis

  4. Version published to 10.64898/2026.03.22.713493 on bioRxiv