Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Matsushita et al. present a review displaying the multiple activities of FIR/PUF60 and their splicing variants. Authors have brought a particular emphasis on the proposed coupling between alternative splicing, transcriptional regulation, ribosome biogenesis, and disease pathogenesis. They have integrated a broad set of literature spanning cancer biology, neurodevelopmental disorders, and spliceosomopathies, while they also included their own published work to combine and propose a conceptual framework in which FIR/PUF60 acts as a pivotal regulator of mRNA and rRNA expression. While the topic is potentially of interest the review frequently blurs the distinction between established findings and speculative models. Moreover several mechanistic descriptions appear over-interpreted relative to the available evidence. As detailed in the other sections of my review, I noted several significant issues regarding the clarity and balance that currently limit the impact and rigor of the manuscript.

Therefore, I am concluding that this review is of interest; however, the authors should improve the structure and clarity of the text in order to be accepted for publication.

This review is of interest; however, the authors should improve the structure and clarity of the text in order to be accepted for publication.

Comments on the Quality of English Language

The manuscript adopts a descriptive approach but it seems to lack some critical distance expected to be provided in a review article. Several portions of the manuscript appear as an accumulation of observations rather than a carefully and organised synthesis of a question. Therefore it is difficult for the reader to distinguish well-established mechanisms from the author-driven speculations. Moreover, the authors have included their own previous studies, which is somehow potentially valuable, but it is not always enough balanced with other work from the literature, making their conclusion too oriented. Several times, the models or conclusions are extrapolated from a limited number of studies and they are presented as a generality or consensus that is not fairly established in the field. Altogether, this writing tends to overstate the strength of the framework and weakens the overall critical rigor of the review. Finally, the manuscript would also benefit from substantial improvements in organization and precision. Together, these issues reduce readability and obscure the main conceptual messages the authors aim to convey.

As outlined in the comments, these points would require not only editorial polishing, but some work from authors to improve their manuscript in order to be more careful in the separation between evidence-based conclusions and speculative interpretations. Such improvement will bring a clearer and more balanced positioning of FIR/PUF60 within the existing landscape of RNA splicing and ribosome biogenesis research.

 

Major comments

 

1. Insufficient distinction between evidence and speculation

Throughout the manuscript, the authors should distinguish between findings and speculative models by making a clear separation and explanation between these two sets. Same should be added to the proposed mechanisms which are over-interpreted relative to the available evidence and would require a more cautious framing.

 

1. The conclusions are too much generalized, often from a limited amount of studies

Many conclusions are claimed from a small number of studies, often originating from the authors’ own work, which they next extended to broad biological contexts without making sufficient moderation in their discussion, for instance by presenting the experimental limitations or alternative interpretations. A more balanced integration of independent literature is needed.

 

1. Conceptual and mechanistic ambiguities

The author’s claims regarding RNAPII playing a role in rRNA transcription, FIR/PUF60–TFIIH interactions, and the functional consequences of this coupling mechanisms are not sufficiently supported by current knowledge. I am proposing that authors improve these sections which would benefit from clearer definitions, stronger contextualization, and a more critical discussion of uncertainties.

 

1. Figures and overall organization

Several figures lack sufficient precision and do not clearly distinguish between established mechanisms and hypothetical or speculative models. In addition, the overall organization of the manuscript would benefit from substantial restructuring to improve clarity and narrative coherence. Figures are not always integrated into the text in a reader-friendly manner, making it difficult to follow how they support the corresponding arguments. Furthermore, multiple errors are present in the figure labels and legends; for example, Figure 2 contains a typographical error (“transcroption” instead of “transcription”), which reflects a lack of careful editing and detracts from the overall quality of the presentation.

Author Response

Reviewer1：　Comments and Suggestions for Authors

In this manuscript, Matsushita et al. present a review displaying the multiple activities of FIR/PUF60 and their splicing variants. Authors have brought a particular emphasis on the proposed coupling between alternative splicing, transcriptional regulation, ribosome biogenesis, and disease pathogenesis. They have integrated a broad set of literature spanning cancer biology, neurodevelopmental disorders, and spliceosomopathies, while they also included their own published work to combine and propose a conceptual framework in which FIR/PUF60 acts as a pivotal regulator of mRNA and rRNA expression. While the topic is potentially of interest the review frequently blurs the distinction between established findings and speculative models. Moreover several mechanistic descriptions appear over-interpreted relative to the available evidence. As detailed in the other sections of my review, I noted several significant issues regarding the clarity and balance that currently limit the impact and rigor of the manuscript.

Reply: All the authors appreciate for accurately reading the gist of our review paper. As you pointed out, we have revised it by dividing it into experimental facts and matters for which there is little data to verify. In particular, the mechanism by which FIR/PUF60 is involved in neural differentiation during childhood development and cell carcinogenesis in adulthood requires much further research, so we have summarized and added a separate section, 6.4. Future issues and challenges to be addressed (lines 606-666).

Therefore, I am concluding that this review is of interest; however, the authors should improve the structure and clarity of the text in order to be accepted for publication.

This review is of interest; however, the authors should improve the structure and clarity of the text in order to be accepted for publication.

Reply: The authors appreciate for your positive and constructive review of our paper. We have revised the paper in line with the points you made. We appreciate you taking the time out of your busy schedule to review it.

Comments on the Quality of English Language

The manuscript adopts a descriptive approach but it seems to lack some critical distance expected to be provided in a review article. Several portions of the manuscript appear as an accumulation of observations rather than a carefully and organized synthesis of a question. Therefore, it is difficult for the reader to distinguish well-established mechanisms from the author-driven speculations. Moreover, the authors have included their own previous studies, which is somehow potentially valuable, but it is not always enough balanced with other work from the literature, making their conclusion too oriented. Several times, the models or conclusions are extrapolated from a limited number of studies and they are presented as a generality or consensus that is not fairly established in the field. Altogether, this writing tends to overstate the strength of the framework and weakens the overall critical rigor of the review. Finally, the manuscript would also benefit from substantial improvements in organization and precision. Together, these issues reduce readability and obscure the main conceptual messages the authors aim to convey.

As outlined in the comments, these points would require not only editorial polishing, but some work from authors to improve their manuscript in order to be more careful in the separation between evidence-based conclusions and speculative interpretations. Such improvement will bring a clearer and more balanced positioning of FIR/PUF60 within the existing landscape of RNA splicing and ribosome biogenesis research.

 Reply: Thank you for taking the time to provide your detailed and accurate feedback. Following your advice, I have tried to be more careful in distinguishing between evidence-based conclusions and speculative interpretations. Regarding the specific work of the authors, I have responded to each of the points below.

Major comments

 

1. Insufficient distinction between evidence and speculation

Throughout the manuscript, the authors should distinguish between findings and speculative models by making a clear separation and explanation between these two sets. Same should be added to the proposed mechanisms which are over-interpreted relative to the available evidence and would require a more cautious framing.

Reply: In addition, at the request of the editorial department, we have briefly revised the following sections.

1.During our review, we noted that some sentences in the manuscript bear similarity to passages in previously published works. To ensure the originality and integrity of the research, we kindly invite you to review and revise the following sections: Lines 106-124；Lines 238-259；Lines 260-270；Lines 272-282；Lines 621-626；Lines 581-610；Lines 289-306.

 

 Reply: Thank you for your accurate comments. I have deleted redundant and overlapping sentences as suggested.

Next paragraph of the last part in the abstract (lines 42-45) was moved to the Discussion (lines 740-743).

This special issue focuses on aberrant splicing and presents promising new findings on disease-related protein diversity of FIR/PUF60, novel cell signaling targets, and mechanisms for effective individualized therapies in human diseases that may lead to future diagnostic and therapeutic applications.

Also, I have summarized the speculations that are not based on experimental data in 6.4. Future issues and challenges to be addressed. I have left a record of the deleted parts in ijms-4060404ver2. Lines 92-95 were deleted.

In humans, three RNA polymerases (RNAPs) are essential for RNA synthesis: RNAPI for rRNA, RNAPII for mRNA, and RNAPIII for tRNA. rRNA is transcribed from ribosomal DNA (rDNA) or rRNA genes in the nucleolus, and the RPs that constitute the ribosome are generated.

Line 97 was deleted.

Thus, RPB6 is a key molecule for integrating mRNA and rRNA transcription.

Lines 104-106 was deleted.

In human diseases, fusion between these acrocentric chromosomes results in Robertsonian translocations (RTs), leading to the loss of the short arms containing 47S rDNA [7].

Lines 239-247 was deleted.

Regarding the participation of FIR in dynamic rRNA transcription, the effective accessibility of FIR to common transcription factors on rDNA is important (Figure 4). The rDNAs affected by the overexpression or knockdown of FIR and FIRΔexon2 were located on chromosomes 17, 12, 19, 5, 2, 6, 9, and 1. These results indicated that FIR and FIRΔexon2 targeted nucleolar rRNA genes on specific chromosomes other than the acrocentric chromosomes 13–15, 21, and 22. Collectively, these results indicated that RNAPII operated directly on rRNA genes [5] in human nucleoli, and that FIR and FIRΔexon2 potentially affected nucleolar rRNA expression, at least in part, via RNAPII.

Lines 249-251 was deleted.

However, the findings of this study strongly suggested that FIR and FIRΔexon2 activated RP genes or rDNAs on specific chromosomes other than the five acrocentric chromosomes.

Lines 253-259 was deleted.

Thus, the shift in expression from FIR to FIRΔexon2 was responsible for aberrant rRNA transcription in cancer. Together, FIR and FIRΔexon2 notably generated rRNA expression of various RPs on chromosomes 17, 12, 19, and 1. This suggested that FIR and FIRΔexon2 stimulated nucleolar rDNA or genes for rRNA transcription on specific chromosomes, in addition to acrocentric chromosomes 13, 14, 15, 21, and 22. Therefore, it was speculated that the common regulatory pathway of mRNA and rRNA by FIR interacted directly with the P62 PH domain of TFIIH, which binds to RPB6 [16].　

Lines 274-279 was deleted.

In intractable diseases characterized by insufficient rRNA synthesis, excluding cancer, germline variants were detected at the RRM1 and RRM2 sites required for FIR dimerization, indicating that FIR dimerization is essential for rRNA synthesis [16]. PUF60 exon 5 is fundamental to the regulation of neurogenesis via a mutually exclusive alternative splicing switch from PUF60 to FIR, remodeling splicing cofactor interactions [2].

Lines 290-293 was deleted.

thereby offering a clue regarding a therapeutic target." These results have elucidated a novel mechanism indicating that FIRΔexon2 regulates the dynamic expression of rRNA in cancer. Specifically, FIR binds to P62 in noncancerous conditions, maintaining RNAPI/II in a stable state.

Lines 299-302 was deleted.

In other words, in noncancerous cells, FIR suppresses rRNA and mRNA transcription by inhibiting the interaction between P62 and RBP6/RNAPI/II. However, in cancerous cells, FIRΔexon2 prevents FIR from binding to P62 and promotes the interaction between P62 and the RPB6 subunit of RNAPI/II, driving rRNA and mRNA transcription.

Lines 588-590 was deleted.

The in vivo antitumor therapeutic efficacy of a replication-defective adenovirus (E1- and E3-deleted adenovirus, serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in a tumor xenograft model using the human ESCC line TE-2.

Lines 592-597 was deleted.

To examine the effect of Ad-FIR on DNA damage repair, BLM, X-rays, and carbon ion irradiation were used as DNA-damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy, such as carbon ion irradiation, are more extensive than those of conventional low-LET radiotherapy, such as X-rays or γ-rays. High-LET radiotherapy is suitable for local tumor control due to its high relative biological effectiveness.

Lines 602-605 was deleted.

SeV/dF/FIR was examined regarding its gene transduction efficiency, the viral dose-dependence of its antitumor effect, and apoptosis induction in HeLa cells (cervical squamous cell carcinoma) and SW480 cells (colon adenocarcinoma). Antitumor efficacy in a mouse xenograft model was also examined.

Lines 608-610 was deleted.

SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and demonstrated highly efficient gene transduction in in vivo experiments.

Lines 623-625 was deleted.

. BK697, which targets FIRΔexon2, may serve as a potential anticancer agent by suppressing rRNA and mRNA transcription through the promotion of the FIR-P62 interaction.

Lines 730-759 was deleted.　In discussion.

Common Transcriptional Activation Mechanisms include:

1. Activation of the mTOR signaling pathway

• mTORC1 is a central pathway that affects both RNA polymerase I (rRNA) and RNA polymerase II (mRNA).
• It promotes rRNA synthesis in the nucleolus and simultaneously activates the transcription of RP genes involved in mRNA translation.
• Increases cellular protein synthesis capacity, depending on nutritional status and growth factors.

2. MYC overexpression

• The MYC transcription factor, frequently activated in cancer, activates all three RNA polymerases: I, II, and III.
• It directly promotes the transcription of rRNA genes and simultaneously increases the transcription of mRNAs related to the cell cycle and metabolism.
• MYC is also involved in reorganizing nucleolar structure, accelerating ribosome biogenesis.

3. Epigenetic chromatin remodeling

• Histone modifications (e.g., H3K4me3, H3K27ac) establish an open chromatin state at the rDNA region and mRNA gene regions.
• This facilitates access by chromatin remodeling factors (e.g., SWI/SNF) to the transcription start sites of both.

4. Regulation by metabolites (e.g., acetyl-CoA, SAM)

• Changes in cancer metabolism lead to increased levels of acetyl-CoA and S-adenosylmethionine (SAM), promoting histone acetylation and methylation.
• This simultaneously increases rRNA and mRNA transcriptional activity. Supplement: Crosstalk between the Nucleolus and Transcription Machinery.
• rRNA is transcribed in the nucleolus by RNA polymerase I, whereas mRNA is transcribed in the nucleoplasm by RNA polymerase II, but the activity of both is coordinated in response to the cell cycle and growth signals.
• In particular, the coordinated transcription of RP genes (mRNA) and rRNA genes ensures a balanced supply of ribosomal components.

 

1. The conclusions are too much generalized, often from a limited amount of studies

Many conclusions are claimed from a small number of studies, often originating from the authors’ own work, which they next extended to broad biological contexts without making sufficient moderation in their discussion, for instance by presenting the experimental limitations or alternative interpretations. A more balanced integration of independent literature is needed.

  Reply: Thank you for pointing this out. I have minimized and removed the in-text description of the speculative model (as mentioned above), and summarized the experimental limitations and alternative interpretations in a separate section, 6.4. Future issues and challenges to be addressed (lines 606-666).

Conceptual and mechanistic ambiguities

The author’s claims regarding RNAPII playing a role in rRNA transcription, FIR/PUF60–TFIIH interactions, and the functional consequences of this coupling mechanisms are not sufficiently supported by current knowledge. I am proposing that authors improve these sections which would benefit from clearer definitions, stronger contextualization, and a more critical discussion of uncertainties.

  Reply:　Next sentences, lines 670-699, were deleted in Discussion and revised in 6.4. Future issues and challenges to be addressed in pages 16-17.　

Common Transcriptional Activation Mechanisms include:

1. Activation of the mTOR signaling pathway

• mTORC1 is a central pathway that affects both RNA polymerase I (rRNA) and RNA polymerase II (mRNA).
• It promotes rRNA synthesis in the nucleolus and simultaneously activates the transcription of RP genes involved in mRNA translation.
• Increases cellular protein synthesis capacity, depending on nutritional status and growth factors.

2. MYC overexpression

• The MYC transcription factor, frequently activated in cancer, activates all three RNA polymerases: I, II, and III.
• It directly promotes the transcription of rRNA genes and simultaneously increases the transcription of mRNAs related to the cell cycle and metabolism.
• MYC is also involved in reorganizing nucleolar structure, accelerating ribosome biogenesis.

3. Epigenetic chromatin remodeling

• Histone modifications (e.g., H3K4me3, H3K27ac) establish an open chromatin state at the rDNA region and mRNA gene regions.
• This facilitates access by chromatin remodeling factors (e.g., SWI/SNF) to the transcription start sites of both.

4. Regulation by metabolites (e.g., acetyl-CoA, SAM)

• Changes in cancer metabolism lead to increased levels of acetyl-CoA and S-adenosylmethionine (SAM), promoting histone acetylation and methylation.
• This simultaneously increases rRNA and mRNA transcriptional activity. Supplement: Crosstalk between the Nucleolus and Transcription Machinery.
• rRNA is transcribed in the nucleolus by RNA polymerase I, whereas mRNA is transcribed in the nucleoplasm by RNA polymerase II, but the activity of both is coordinated in response to the cell cycle and growth signals.
• In particular, the coordinated transcription of RP genes (mRNA) and rRNA genes ensures a balanced supply of ribosomal components.

6.4. Future issues and challenges to be addressed

The mechanisms involved in human diseases include splicing, rRNA-mRNA coupling, non-coding or coding RNA transcription and ORF-dominancy, and abnormalities in ribosome biogenesis. Although these are all interrelated, their relationship to disease and their details remain largely unknown, as described below.

1) Elucidation of the common activation mechanism of rRNA and mRNA during neural differentiation and carcinogenesis. Simultaneous transcriptional activation of rRNA and mRNA in cancer cells requires a rapid increase in protein synthesis capacity necessary for cell proliferation, suggesting coordinated regulation of nucleolar function and RNA polymerase activity.

1. Activation of the mTOR signaling pathway
2. Overexpression of MYC
3. Epigenetic chromatin remodeling

2) FIR/PUF60 is involved in cancer cell proliferation, metabolism, and survival through regulation of RNA splicing and mRNA stability. Meanwhile, mTOR signaling functions as an upstream regulator of translation, metabolism, and the cell cycle. Future research will clarify whether PUF60 alters splicing patterns depending on the activation state of the mTOR pathway, or whether mTOR inhibition affects PUF60 function. The hypothesis that FIR/PUF60 regulates translation efficiency through splicing control is an intriguing topic for elucidating the molecular mechanisms of cancer and metabolic diseases.

3) How is unified transcription of human rDNA possible? What is the connection between ribosomal abnormalities and splicing abnormalities?

1. Shared and Interfered Nuclear Structures: Ribosome biogenesis occurs in the nucleolus, and splicing occurs in nuclear speckles. Reorganization of the nuclear structure due to nucleolar stress may also affect the localization and function of splicing factors.
2. Multifunctionality of Ribosomal Proteins
3. Coordinated Regulation of Translation and Splicing:Recent studies have suggested that translational regulation and splicing regulation are linked, and that ribosomal abnormalities may affect the selection of mRNA splice variants. In particular, the stress response associated with ribosomal pathologies can lead to changes in the expression and modification of splicing factors.

4) Main Reasons Why Abnormal Splicing Causes Ribosomal Abnormalities

1. Abnormal mRNA Splicing of Ribosomal Components. Abnormal splicing of pre-mRNAs encoding components such as ribosomal proteins (RPs) and rRNA-modifying enzymes results in a decrease in translatable mRNAs or the accumulation of abnormal isoforms.
2. Impaired rRNA Processing Due to Abnormal snRNP Function. Mutations in spliceosome components (e.g., SF3B1, U2AF1) also affect pre-rRNA processing through snRNP dysfunction. Accurate cleavage and modification of rRNA are inhibited, resulting in incomplete formation of mature ribosomes.
3. Disrupted Nucleolar Function. Abnormal splicing alters the expression and localization of nucleolar-localized proteins. Because the nucleolus is the site of rRNA transcription, processing, and ribosome assembly, changes in nucleolar structure are directly linked to ribosome formation.
4. Decreased rDNA Transcription Due to Impaired Transcription Elongation. rDNA transcription is Pol I-dependent, but it has been reported that intranuclear crosstalk also suppresses Pol I activity, resulting in reduced rRNA production. Investigating the involvement of FIR/PUF60 splicing in these mechanisms is a future challenge that will provide new insights into the mechanisms of neuronal differentiation and oncogenesis.

6.5. Limitations

1) The mechanism and the specific d proteins described in this review are limited and require further identification.

2) The interprepation among intermolecular crosstalk are complicated is not well supported by current knowledge:

1. Abnormal splicing → Abnormal mRNA → Abnormal translation → Ribosomal stress.
2. Ribosomal abnormalities → Altered translation selectivity → Altered expression and modification of splicing factors
3. Functional interference due to rearrangements of intranuclear structures (nucleoli and speckles)

 

1. Figures and overall organization

Several figures lack sufficient precision and do not clearly distinguish between established mechanisms and hypothetical or speculative models. In addition, the overall organization of the manuscript would benefit from substantial restructuring to improve clarity and narrative coherence. Figures are not always integrated into the text in a reader-friendly manner, making it difficult to follow how they support the corresponding arguments. Furthermore, multiple errors are present in the figure labels and legends; for example, Figure 2 contains a typographical error (“transcroption” instead of “transcription”), which reflects a lack of careful editing and detracts from the overall quality of the presentation.

 Reply: Following the editorial staff's advice, the order of the figures has been changed as shown below. Accordingly, the figure numbers in the text and legends have also been revised.

Figure 1→３. The necessary information has been added to the figures, and Figures 1, 2A/B, and 4 have been replaced.

Figure ２→1

Figure 3→8

Figure 4→2

Figure 5→4

Figure 6→３

Figure 7→8

Figure 8→6

Figure 9→7

 

(To Editor) We noted that some information is currently missing from the back matter of your manuscript. Please ensure it is completed in the revised version.

 Reply: I have filled in the back matter section of the paper.

Minor points

1. Reply: The order of the figures has been changed and rearranged in accordance with the editorial staff's suggestions.
2. The reference numbers have been rearranged in accordance with the deletion and addition of duplicated sections of the main text and the change in the order of figures.
3. Typographical errors in the figures have been corrected, and B and C have been added to Figure 2. These figures relate to TFIIH interaction and rRNA/mRNA regulation.

 

Submission Date

08 December 2025

Date of this review

18 Dec 2025 16:30:19

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The revisions made by the authors significantly improved the readability and the overall quality of the manuscript. The review became eligible for publication in International Journal of Molecular Science

Author Response

Reviewer2：　Comments and Suggestions for Authors

The revisions made by the authors significantly improved the readability and the overall quality of the manuscript. The review became eligible for publication in International Journal of Molecular Science

Submission Date

08 December 2025

Date of this review

26 Dec 2025 11:50:56

Reply: We would like to thank you for taking the time out of your busy schedule to review this paper, and we would like to thank you for your supportive comments on this paper.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive and ambitious review of FIR/PUF60 biology, focusing on its roles in RNA splicing, transcriptional regulation, ribosome biogenesis, ORF dominance, and disease pathogenesis, particularly cancer and neurodevelopmental disorders. The authors integrate a large body of experimental and clinical literature and propose a unifying framework in which FIR/PUF60 splicing variants coordinate mRNA and rRNA transcription via TFIIH, ribosomal pathways, and chromatin remodeling.

The review is rich in mechanistic detail and will be valuable to specialists. However, the manuscript is overly long, dense, and difficult to follow in its current form, and significant restructuring and clarification are required to improve readability, coherence, and impact.

Length and Organization

• The manuscript is excessively long for a review article and contains substantial repetition, particularly regarding:
• FIRΔexon2 dominant-negative effects
• c-MYC-dependent vs. independent mechanisms
• TFIIH–P62–RPB6 interactions
• Several sections (e.g., ribosome biogenesis, ORF dominance, longevity/immunity) would benefit from condensation or clearer prioritization.

Recommendation:
Substantially shorten the manuscript by:

• Removing redundant explanations
• Consolidating overlapping mechanistic descriptions
• Focusing on the most novel and unifying concepts (e.g., FIR-mediated coupling of rRNA and mRNA transcription)

Conceptual Clarity and Central Message

• While the review covers many biological processes, the central conceptual model is sometimes obscured by excessive detail.
• The proposed c-MYC-independent role of FIRΔexon2 in regulating rRNA/mRNA transcription is intriguing but not always clearly separated from c-MYC-dependent mechanisms.

Recommendation:

• Clearly define and emphasize:
• What is established knowledge
• What is the authors’ integrative interpretation
• What remains speculative
• A summary schematic or a concise “Key Concepts” box early in the manuscript would greatly improve clarity.

ORF Dominance Section

• The discussion of ORF dominance is intellectually interesting and potentially novel, but it is very long and may be difficult for readers unfamiliar with the concept.
• The biological relevance of ORF dominance to FIR/PUF60-driven disease phenotypes is not always explicit.

Recommendation:

• Clarify how ORF dominance directly informs disease mechanisms or therapeutic strategies

Clinical Relevance

• • The biomarker and therapeutic sections are promising but could be better structured to clearly separate:
    • Diagnostic potential
    • Therapeutic strategies
    • Preclinical vs. clinical evidence
  • Figures
• Arrange the figures properly and in order. 
• Consider adding a final “Graphical Summary” figure integrating FIR/PUF60 splicing, TFIIH interaction, rRNA/mRNA regulation, and disease outcomes.

Author Response

Reviewer3：　Comments and Suggestions for Authors

This manuscript presents a comprehensive and ambitious review of FIR/PUF60 biology, focusing on its roles in RNA splicing, transcriptional regulation, ribosome biogenesis, ORF dominance, and disease pathogenesis, particularly cancer and neurodevelopmental disorders. The authors integrate a large body of experimental and clinical literature and propose a unifying framework in which FIR/PUF60 splicing variants coordinate mRNA and rRNA transcription via TFIIH, ribosomal pathways, and chromatin remodeling.

Reply: I would like to thank you for your thorough review of this paper and for your constructive advice and insightful comments. I have made revisions based on your suggestions.

The review is rich in mechanistic detail and will be valuable to specialists. However, the manuscript is overly long, dense, and difficult to follow in its current form, and significant restructuring and clarification are required to improve readability, coherence, and impact.

Reply: In accordance with the reviewer's comments, we have tried to critically describe overlapping sections, concepts with little experimental data, and concepts that are not yet established. Therefore, we have deleted the descriptions in the main text and summarized them in 6.4. Future issues and challenges to be addressed in pages 16-17.

 

Length and Organization

• The manuscript is excessively long for a review article and contains substantial repetition, particularly regarding:
• FIRΔexon2 dominant-negative effects
• c-MYC-dependent vs. independent mechanisms
• TFIIH–P62–RPB6 interactions
• Several sections (e.g., ribosome biogenesis, ORF dominance, longevity/immunity) would benefit from condensation or clearer prioritization.

Reply: As the reviewer pointed out, the purpose of this paper is threefold. Although the number of papers on PUF60/FIR has been increasing in recent years, there are still many unclear points due to the small number of researchers. We hope that this review will contribute to the advancement of research. Regarding the TFIIH–P62–RPB6 interactions, we added new figures to Figure 2B and C as suggested by the reviewers, as the description alone was difficult to understand.　Also, the authors shorten the length of the manuscript after revision.

 

Recommendation:
Substantially shorten the manuscript by:

• Removing redundant explanations
• Consolidating overlapping mechanistic descriptions
• Focusing on the most novel and unifying concepts (e.g., FIR-mediated coupling of rRNA and mRNA transcription)

Reply: Following reviewer suggestions, we have removed redundant and repetitive statements, and added new figures to Figure 2B and C to explain FIR-mediated coupling of rRNA and mRNA transcription.

 

Thank you for your accurate comments. I have deleted redundant and overlapping sentences as suggested. Also, I have summarized the speculations that are not based on experimental data in 6.4. Future issues and challenges to be addressed. I have left a record of the deleted parts in ijms-4060404ver2. Lines 92-95 were deleted.

In humans, three RNA polymerases (RNAPs) are essential for RNA synthesis: RNAPI for rRNA, RNAPII for mRNA, and RNAPIII for tRNA. rRNA is transcribed from ribosomal DNA (rDNA) or rRNA genes in the nucleolus, and the RPs that constitute the ribosome are generated.

Line 97 was deleted.

Thus, RPB6 is a key molecule for integrating mRNA and rRNA transcription.

Lines 104-106 was deleted.

In human diseases, fusion between these acrocentric chromosomes results in Robertsonian translocations (RTs), leading to the loss of the short arms containing 47S rDNA [7].

Lines 239-247 was deleted.

Regarding the participation of FIR in dynamic rRNA transcription, the effective accessibility of FIR to common transcription factors on rDNA is important (Figure 4). The rDNAs affected by the overexpression or knockdown of FIR and FIRΔexon2 were located on chromosomes 17, 12, 19, 5, 2, 6, 9, and 1. These results indicated that FIR and FIRΔexon2 targeted nucleolar rRNA genes on specific chromosomes other than the acrocentric chromosomes 13–15, 21, and 22. Collectively, these results indicated that RNAPII operated directly on rRNA genes [5] in human nucleoli, and that FIR and FIRΔexon2 potentially affected nucleolar rRNA expression, at least in part, via RNAPII.

Lines 249-251 was deleted.

However, the findings of this study strongly suggested that FIR and FIRΔexon2 activated RP genes or rDNAs on specific chromosomes other than the five acrocentric chromosomes.

Lines 253-259 was deleted.

Thus, the shift in expression from FIR to FIRΔexon2 was responsible for aberrant rRNA transcription in cancer. Together, FIR and FIRΔexon2 notably generated rRNA expression of various RPs on chromosomes 17, 12, 19, and 1. This suggested that FIR and FIRΔexon2 stimulated nucleolar rDNA or genes for rRNA transcription on specific chromosomes, in addition to acrocentric chromosomes 13, 14, 15, 21, and 22. Therefore, it was speculated that the common regulatory pathway of mRNA and rRNA by FIR interacted directly with the P62 PH domain of TFIIH, which binds to RPB6 [16].　

Lines 274-279 was deleted.

In intractable diseases characterized by insufficient rRNA synthesis, excluding cancer, germline variants were detected at the RRM1 and RRM2 sites required for FIR dimerization, indicating that FIR dimerization is essential for rRNA synthesis [16]. PUF60 exon 5 is fundamental to the regulation of neurogenesis via a mutually exclusive alternative splicing switch from PUF60 to FIR, remodeling splicing cofactor interactions [2].

Lines 290-293 was deleted.

thereby offering a clue regarding a therapeutic target." These results have elucidated a novel mechanism indicating that FIRΔexon2 regulates the dynamic expression of rRNA in cancer. Specifically, FIR binds to P62 in noncancerous conditions, maintaining RNAPI/II in a stable state.

Lines 299-302 was deleted.

In other words, in noncancerous cells, FIR suppresses rRNA and mRNA transcription by inhibiting the interaction between P62 and RBP6/RNAPI/II. However, in cancerous cells, FIRΔexon2 prevents FIR from binding to P62 and promotes the interaction between P62 and the RPB6 subunit of RNAPI/II, driving rRNA and mRNA transcription.

Lines 588-590 was deleted.

The in vivo antitumor therapeutic efficacy of a replication-defective adenovirus (E1- and E3-deleted adenovirus, serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in a tumor xenograft model using the human ESCC line TE-2.

Lines 592-597 was deleted.

To examine the effect of Ad-FIR on DNA damage repair, BLM, X-rays, and carbon ion irradiation were used as DNA-damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy, such as carbon ion irradiation, are more extensive than those of conventional low-LET radiotherapy, such as X-rays or γ-rays. High-LET radiotherapy is suitable for local tumor control due to its high relative biological effectiveness.

Lines 602-605 was deleted.

SeV/dF/FIR was examined regarding its gene transduction efficiency, the viral dose-dependence of its antitumor effect, and apoptosis induction in HeLa cells (cervical squamous cell carcinoma) and SW480 cells (colon adenocarcinoma). Antitumor efficacy in a mouse xenograft model was also examined.

Lines 608-610 was deleted.

SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and demonstrated highly efficient gene transduction in in vivo experiments.

Lines 623-625 was deleted.

. BK697, which targets FIRΔexon2, may serve as a potential anticancer agent by suppressing rRNA and mRNA transcription through the promotion of the FIR-P62 interaction.

Lines 730-759 was deleted.　In discussion.

Common Transcriptional Activation Mechanisms include:

1. Activation of the mTOR signaling pathway

• mTORC1 is a central pathway that affects both RNA polymerase I (rRNA) and RNA polymerase II (mRNA).
• It promotes rRNA synthesis in the nucleolus and simultaneously activates the transcription of RP genes involved in mRNA translation.
• Increases cellular protein synthesis capacity, depending on nutritional status and growth factors.

2. MYC overexpression

• The MYC transcription factor, frequently activated in cancer, activates all three RNA polymerases: I, II, and III.
• It directly promotes the transcription of rRNA genes and simultaneously increases the transcription of mRNAs related to the cell cycle and metabolism.
• MYC is also involved in reorganizing nucleolar structure, accelerating ribosome biogenesis.

3. Epigenetic chromatin remodeling

• Histone modifications (e.g., H3K4me3, H3K27ac) establish an open chromatin state at the rDNA region and mRNA gene regions.
• This facilitates access by chromatin remodeling factors (e.g., SWI/SNF) to the transcription start sites of both.

4. Regulation by metabolites (e.g., acetyl-CoA, SAM)

• Changes in cancer metabolism lead to increased levels of acetyl-CoA and S-adenosylmethionine (SAM), promoting histone acetylation and methylation.
• This simultaneously increases rRNA and mRNA transcriptional activity. Supplement: Crosstalk between the Nucleolus and Transcription Machinery.
• rRNA is transcribed in the nucleolus by RNA polymerase I, whereas mRNA is transcribed in the nucleoplasm by RNA polymerase II, but the activity of both is coordinated in response to the cell cycle and growth signals.
• In particular, the coordinated transcription of RP genes (mRNA) and rRNA genes ensures a balanced supply of ribosomal components.

 

1. The conclusions are too much generalized, often from a limited amount of studies

Many conclusions are claimed from a small number of studies, often originating from the authors’ own work, which they next extended to broad biological contexts without making sufficient moderation in their discussion, for instance by presenting the experimental limitations or alternative interpretations. A more balanced integration of independent literature is needed.

  Reply: All the authors appreciate for accurately reading the gist of our review paper. The authors agreed the points that the reviewer recommended to revise. As the reviewer pointed out, we have revised it by dividing it into experimental facts and matters for which there is little data to verify. In particular, the mechanism by which FIR/PUF60 is involved in neural differentiation during childhood development and cell carcinogenesis in adulthood requires much further research, so we have summarized and added a separate section, 6.4. Future issues and challenges to be addressed (lines 606-666).

 

1. Conceptual and mechanistic ambiguities

The author’s claims regarding RNAPII playing a role in rRNA transcription, FIR/PUF60–TFIIH interactions, and the functional consequences of this coupling mechanisms are not sufficiently supported by current knowledge. I am proposing that authors improve these sections which would benefit from clearer definitions, stronger contextualization, and a more critical discussion of uncertainties.

  Reply:　Next sentences, lines 670-699, were deleted in Discussion and revised in 6.4. Future issues and challenges to be addressed in pages 16-17.　

Common Transcriptional Activation Mechanisms include:

1. Activation of the mTOR signaling pathway

• mTORC1 is a central pathway that affects both RNA polymerase I (rRNA) and RNA polymerase II (mRNA).
• It promotes rRNA synthesis in the nucleolus and simultaneously activates the transcription of RP genes involved in mRNA translation.
• Increases cellular protein synthesis capacity, depending on nutritional status and growth factors.

2. MYC overexpression

• The MYC transcription factor, frequently activated in cancer, activates all three RNA polymerases: I, II, and III.
• It directly promotes the transcription of rRNA genes and simultaneously increases the transcription of mRNAs related to the cell cycle and metabolism.
• MYC is also involved in reorganizing nucleolar structure, accelerating ribosome biogenesis.

3. Epigenetic chromatin remodeling

• Histone modifications (e.g., H3K4me3, H3K27ac) establish an open chromatin state at the rDNA region and mRNA gene regions.
• This facilitates access by chromatin remodeling factors (e.g., SWI/SNF) to the transcription start sites of both.

4. Regulation by metabolites (e.g., acetyl-CoA, SAM)

• Changes in cancer metabolism lead to increased levels of acetyl-CoA and S-adenosylmethionine (SAM), promoting histone acetylation and methylation.
• This simultaneously increases rRNA and mRNA transcriptional activity. Supplement: Crosstalk between the Nucleolus and Transcription Machinery.
• rRNA is transcribed in the nucleolus by RNA polymerase I, whereas mRNA is transcribed in the nucleoplasm by RNA polymerase II, but the activity of both is coordinated in response to the cell cycle and growth signals.
• In particular, the coordinated transcription of RP genes (mRNA) and rRNA genes ensures a balanced supply of ribosomal components.

6.4. Future issues and challenges to be addressed

The mechanisms involved in human diseases include splicing, rRNA-mRNA coupling, non-coding or coding RNA transcription and ORF-dominancy, and abnormalities in ribosome biogenesis. Although these are all interrelated, their relationship to disease and their details remain largely unknown, as described below.

1) Elucidation of the common activation mechanism of rRNA and mRNA during neural differentiation and carcinogenesis. Simultaneous transcriptional activation of rRNA and mRNA in cancer cells requires a rapid increase in protein synthesis capacity necessary for cell proliferation, suggesting coordinated regulation of nucleolar function and RNA polymerase activity.

1. Activation of the mTOR signaling pathway
2. Overexpression of MYC
3. Epigenetic chromatin remodeling

2) FIR/PUF60 is involved in cancer cell proliferation, metabolism, and survival through regulation of RNA splicing and mRNA stability. Meanwhile, mTOR signaling functions as an upstream regulator of translation, metabolism, and the cell cycle. Future research will clarify whether PUF60 alters splicing patterns depending on the activation state of the mTOR pathway, or whether mTOR inhibition affects PUF60 function. The hypothesis that FIR/PUF60 regulates translation efficiency through splicing control is an intriguing topic for elucidating the molecular mechanisms of cancer and metabolic diseases.

3) How is unified transcription of human rDNA possible? What is the connection between ribosomal abnormalities and splicing abnormalities?

1. Shared and Interfered Nuclear Structures: Ribosome biogenesis occurs in the nucleolus, and splicing occurs in nuclear speckles. Reorganization of the nuclear structure due to nucleolar stress may also affect the localization and function of splicing factors.
2. Multifunctionality of Ribosomal Proteins
3. Coordinated Regulation of Translation and Splicing:Recent studies have suggested that translational regulation and splicing regulation are linked, and that ribosomal abnormalities may affect the selection of mRNA splice variants. In particular, the stress response associated with ribosomal pathologies can lead to changes in the expression and modification of splicing factors.

4) Main Reasons Why Abnormal Splicing Causes Ribosomal Abnormalities

1. Abnormal mRNA Splicing of Ribosomal Components. Abnormal splicing of pre-mRNAs encoding components such as ribosomal proteins (RPs) and rRNA-modifying enzymes results in a decrease in translatable mRNAs or the accumulation of abnormal isoforms.
2. Impaired rRNA Processing Due to Abnormal snRNP Function. Mutations in spliceosome components (e.g., SF3B1, U2AF1) also affect pre-rRNA processing through snRNP dysfunction. Accurate cleavage and modification of rRNA are inhibited, resulting in incomplete formation of mature ribosomes.
3. Disrupted Nucleolar Function. Abnormal splicing alters the expression and localization of nucleolar-localized proteins. Because the nucleolus is the site of rRNA transcription, processing, and ribosome assembly, changes in nucleolar structure are directly linked to ribosome formation.
4. Decreased rDNA Transcription Due to Impaired Transcription Elongation. rDNA transcription is Pol I-dependent, but it has been reported that intranuclear crosstalk also suppresses Pol I activity, resulting in reduced rRNA production. Investigating the involvement of FIR/PUF60 splicing in these mechanisms is a future challenge that will provide new insights into the mechanisms of neuronal differentiation and oncogenesis.

6.5. Limitations

1) The mechanism and the specific d proteins described in this review are limited and require further identification.

2) The interprepation among intermolecular crosstalk are complicated is not well supported by current knowledge:

1. Abnormal splicing → Abnormal mRNA → Abnormal translation → Ribosomal stress.
2. Ribosomal abnormalities → Altered translation selectivity → Altered expression and modification of splicing factors
3. Functional interference due to rearrangements of intranuclear structures (nucleoli and speckles)

Conceptual Clarity and Central Message

• While the review covers many biological processes, the central conceptual model is sometimes obscured by excessive detail.
• The proposed c-MYC-independent role of FIRΔexon2 in regulating rRNA/mRNA transcription is intriguing but not always clearly separated from c-MYC-dependent mechanisms.

Recommendation:

• Clearly define and emphasize:
• What is established knowledge
• What is the authors’ integrative interpretation
• What remains speculative
• A summary schematic or a concise “Key Concepts” box early in the manuscript would greatly improve clarity.

 

Reply: As you pointed out, the authors agree with your opinion. Figure 2B and C show a schematic diagram of FIRΔexon2 in regulating rRNA/mRNA. As the reviewer pointed out, it is difficult to distinguish between c-MYC-independent and c-MYC-dependent roles [5]. This point has been mentioned in the text (lines 248-250) and discussion section (lines 678-681).

ORF Dominance Section

• The discussion of ORF dominance is intellectually interesting and potentially novel, but it is very long and may be difficult for readers unfamiliar with the concept.
• The biological relevance of ORF dominance to FIR/PUF60-driven disease phenotypes is not always explicit.

Recommendation:

• Clarify how ORF dominance directly informs disease mechanisms or therapeutic strategies

Clinical Relevance

• The biomarker and therapeutic sections are promising but could be better structured to clearly separate:
• Diagnostic potential
• Therapeutic strategies
• Preclinical vs. clinical evidence
• Figures
• Arrange the figures properly and in order. 
• Consider adding a final “Graphical Summary” figure integrating FIR/PUF60 splicing, TFIIH interaction, rRNA/mRNA regulation, and disease outcomes.

  Reply:　The authors revised the ORF Dominance Section as follows.

• The discussion of ORF dominance is intellectually interesting and potentially novel, but it is very long and may be difficult for readers unfamiliar with the concept.

 

In accordance with the reviewer's suggestion, we have deleted the details of the circumstances that led to the development of ORF dominance (see the blue text below).

Clarify how ORF dominance directly informs disease mechanisms or therapeutic strategies

In response to this comment, we have added the following three clear biological significances of ORF dominance (see the red text):

Biological Significance of ORF Dominance

1) Extinction and the Emergence of Genes in Biological Evolution

2) Cancer Onset and Progression and the Emergence of Neoantigens

3) Patient Prognosis in Neuroblastoma

（Next paragraph were deleted and revised as follows: ）

 Genes typically produce multiple RNA isoforms, and determining which isoform is actually translated into protein is a fundamental question for understanding disease pathogenesis. Furthermore, noncoding RNAs (ncRNAs) have been found to contain open reading frames (ORFs) that can be translated into functional peptides [98-100], whereas some coding RNAs (mRNAs) exhibit translation-independent regulatory functions [101-103]. For instance, certain known coding RNAs, such as the transcript of the p53 tumor suppressor gene, have been shown to possess physiological functions at the RNA level [104]. Conversely, there are numerous reports of RNAs traditionally classified as noncoding being translated into proteins [98], thereby demonstrating the pathophysiological functions of these proteins [98,99]. These findings have led to the identification of bifunctional RNAs, which possess both coding and noncoding properties. This challenges the conventional dichotomy between coding and noncoding RNAs. Recently, it has been reported that when species face the risk of extinction, the expression patterns of RNA isoforms change, accompanied by an increase in ORF dominance, an index correlated with translational efficiency, thereby promoting the emergence of novel genes [22]. Interestingly, such an increase in ORF dominance is also observed during cancer initiation and progression [98]. Notably, noncoding RNAs that give rise to neoantigens have been shown to exhibit high ORF dominance, suggesting a link between ORF dominance and the generation of immunogenic peptides [98]. In particular, in neuroblastoma, the MYCN oncogene positively regulates and maintains the expression of RNA isoforms with high ORF dominance, and the expression of high ORF dominance transcripts in neuroblastoma is associated with poor patient prognosis [23].

(To Editor) We noted that some information is currently missing from the back matter of your manuscript. Please ensure it is completed in the revised version.

 Reply: I have filled in the back matter section of the paper.

 

Minor points

1. Reply: The order of the figures has been changed and rearranged in accordance with the editorial staff's suggestions.
2. The reference numbers have been rearranged in accordance with the deletion and addition of duplicated sections of the main text and the change in the order of figures.
3. Typographical errors in the figures have been corrected, and B and C have been added to Figure 2. These figures relate to TFIIH interaction and rRNA/mRNA regulation.
4. Next seven referenced were deleted. And the order of remaining referenced were revised in the final manuscript.
   • Broeils LA, Ruiz-Orera J, Snel B, Hubner N, van Heesch S. Evolution and implications of de novo genes in humans. Nat Ecol Evol. 2023 Jun;7(6):804-815. doi: 10.1038/s41559-023-02014-y. Epub 2023 Mar 16. PMID: 36928843.
   • Suenaga Y, Islam SM, Alagu J, Kaneko Y, Kato M, Tanaka Y, Kawana H, Hossain S, Matsumoto D, Yamamoto M, Shoji W, Itami M, Shibata T, Nakamura Y, Ohira M, Haraguchi S, Takatori A, Nakagawara A. NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas. PLoS Genet. 2014 Jan;10(1):e1003996. doi: 10.1371/journal.pgen.1003996. Epub 2014 Jan 2. PMID: 24391509; PMCID: PMC3879166.
   • Zhou X, Qin Y, Li J, Fan L, Zhang S, Zhang B, Wu L, Gao A, Yang Y, Lv X, Guo B, Sun L. LncPepAtlas: a comprehensive resource for exploring the translational landscape of long non-coding RNAs. Nucleic Acids Res. 2025 Jan 6;53(D1):D468-D476.
   • Candeias MM. The can and can't dos of p53 RNA. Biochimie. 2011 Nov;93(11):1962-5. doi: 10.1016/j.biochi.2011.06.010. Epub 2011 Jun 23. PMID: 21722702.
   • Kloc M, Foreman V, Reddy SA. Binary function of mRNA. Biochimie. 2011 Nov;93(11):1955-61. doi: 10.1016/j.biochi.2011.07.008. Epub 2011 Jul 28. PMID: 21784124.
   • Huang Y, Wang J, Zhao Y, Wang H, Liu T, Li Y, Cui T, Li W, Feng Y, Luo J, Gong J, Ning L, Zhang Y, Wang D, Zhang Y. cncRNAdb: a manually curated resource of experimentally supported RNAs with both protein-coding and noncoding function. Nucleic Acids Res. 2021 Jan 8;49(D1):D65-D70. doi: 10.1093/nar/gkaa791. PMID: 33010163; PMCID: PMC7778915.
   • Candeias MM, Malbert-Colas L, Powell DJ, Daskalogianni C, Maslon MM, Naski N, Bourougaa K, Calvo F, Fåhraeus R. P53 mRNA controls p53 activity by managing Mdm2 functions. Nat Cell Biol. 2008 Sep;10(9):1098-105. doi: 10.1038/ncb1770. PMID: 19160491.

 

Submission Date

08 December 2025

Date of this review

26 Dec 2025 00:14:52

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed the point raised during the first round of review. The revised version clarifies this aspect and satisfactorily responds to the reviewer’s concern. In its current form, the manuscript is suitable for publication.

Comments on the Quality of English Language

The manuscript adopts a descriptive approach but it seems to lack some critical distance expected to be provided in a review article. Several portions of the manuscript appear as an accumulation of observations rather than a carefully and organised synthesis of a question. Therefore it is difficult for the reader to distinguish well-established mechanisms from the author-driven speculations. Moreover, the authors have included their own previous studies, which is somehow potentially valuable, but it is not always enough balanced with other work from the literature, making their conclusion too oriented. Several times, the models or conclusions are extrapolated from a limited number of studies and they are presented as a generality or consensus that is not fairly established in the field. Altogether, this writing tends to overstate the strength of the framework and weakens the overall critical rigor of the review. Finally, the manuscript would also benefit from substantial improvements in organization and precision. Together, these issues reduce readability and obscure the main conceptual messages the authors aim to convey.

As outlined in the comments, these points would require not only editorial polishing, but some work from authors to improve their manuscript in order to be more careful in the separation between evidence-based conclusions and speculative interpretations. Such improvement will bring a clearer and more balanced positioning of FIR/PUF60 within the existing landscape of RNA splicing and ribosome biogenesis research.

 

Major comments

 

1. Insufficient distinction between evidence and speculation

Throughout the manuscript, the authors should distinguish between findings and speculative models by making a clear separation and explanation between these two sets. Same should be added to the proposed mechanisms which are over-interpreted relative to the available evidence and would require a more cautious framing.

 

1. The conclusions are too much generalized, often from a limited amount of studies

Many conclusions are claimed from a small number of studies, often originating from the authors’ own work, which they next extended to broad biological contexts without making sufficient moderation in their discussion, for instance by presenting the experimental limitations or alternative interpretations. A more balanced integration of independent literature is needed.

 

1. Conceptual and mechanistic ambiguities

The author’s claims regarding RNAPII playing a role in rRNA transcription, FIR/PUF60–TFIIH interactions, and the functional consequences of this coupling mechanisms are not sufficiently supported by current knowledge. I am proposing that authors improve these sections which would benefit from clearer definitions, stronger contextualization, and a more critical discussion of uncertainties.

 

1. Figures and overall organization

Several figures lack sufficient precision and do not clearly distinguish between established mechanisms and hypothetical or speculative models. In addition, the overall organization of the manuscript would benefit from substantial restructuring to improve clarity and narrative coherence. Figures are not always integrated into the text in a reader-friendly manner, making it difficult to follow how they support the corresponding arguments. Furthermore, multiple errors are present in the figure labels and legends; for example, Figure 2 contains a typographical error (“transcroption” instead of “transcription”), which reflects a lack of careful editing and detracts from the overall quality of the presentation.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have done great in adressing all the comments and suggestions. The review is now suitable for publication. All the best to the authors.