The Inevitable Relationship Between Viruses and RNA Modifications Revealed Through Adenovirus Research

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Comments and Suggestions for Authors

Here are some of my comments regarding the manuscript:

L64-66: The authors state that they will discuss HAdV splicing in the manuscript. However, that is not really done! HAdV splicing is not only about E1A but also about MLTU, where Prof. Akusjärvi's research group has conducted several groundbreaking mechanistic studies. Also, the authors do not provide any mechanistic explanations of E1A splicing, which would be helpful to show the beauty of pre-mRNA splicing in HAdV infections.

L72-74: not the best sentence, as it is not only oncogenes in viruses that are spliced. Take HAdV, all capsid protein mRNAs are spliced, and they are not oncogenes.

L74-75: The authors should use acronyms. Also, what kind of polyomaviruses (PyVs) are the authors referring to? Using Fields “Virology” is a low level of reference usage; there should be more recent reviews on PyVs.

L81: “Boldly” is slang and should not be used in scientific texts.

L100-102: unclear sentences. Is it the author’s plan to study these topics? Also, what do the authors mean by “consider the possibility that siRNAs derived from cellular genes infected by a virus are transported to polyribosomes”?.This has been known for a long time.

L133: MMLV has already been defined as the acronym at the beginning of the text.

L187-191: Well, it is OK to give credit to the original paper, but many more studies have been done during the last 20 years in the miRNA and miRNA target topics. These sentences should be rephrased, and more updated data about miRNAs should be presented.

L214-219: There is no reference to these statements. Fix it.

L302: Why define METTL3 here, as you have used it already before? And it is just not a component of the complex but a catalytic subunit.

L314: ncRNA has been defined already before…

L315-331: This part should be fused to the previous paragraph (L227-240) to avoid redundancy.

L333: not correct statement: small ncRNAs are not only miRNA and siRNA; we do have piRNA, tRFs, rRFs, sdRNAs...

L343: HCV as the acronyme

L344: remove “(the specific name of this miRNA)”

L347: rephrase “miRNAs derived from cellular DNA” as cellular miRNA

L352-353: rephrase sentence “And they are transcribed from adenovirus DNA.” And use it in conjunction with the preceding sentence.

L395: Use proper acronyms

L399: TSS has been defined already above

L406-413. Unclear sentences with a totally irrelevant message, rephrase.

L432-437: too detailed descriptions, it is not a methodology paper. Remove or rephrase it.

L474: What is the meaning of this sentence? Rephrase or remove it

L500-502: Since the authors do not discuss the splicing regulation, these sentences should be removed.

L599-601:Well, I do not agree with these statements. The review so far consists only of random “bits and pieces” that do not lead to a very good, comprehensive understanding of the topic. I suggest removing it.

L612-620: Move it to the main body of the text; it is not a “Conclusion” section, it is rather a technical description.

The title (“The inevitable relationship between viruses and RNA revealed 2 through adenovirus research: Viruses make amazing use of 3 RNA to multiply”) is too ambiguous for this manuscript's quality. It has to be rephrased to reflect the review content. For example, “The inevitable relationship between viruses and RNA modifications revealed through adenovirus research.

Finally, I have not understood why the authors have not focused solely on HAdV and its mRNA modifications by capping (the authors' expertise!), splicing, polyadenylation, and post-transcriptional modifications (e.g., m6A, but the authors obviously have experience in other modifications as well). I personally would have skipped the. ncRNA part. This is the review topic that we really miss in the adenovirus field, with reflections on studies done in the 70s and 80s.... 

 

Comments on the Quality of English Language

Has to be improved, and a REAL English-speaker and someone who can combine sentences and paragraphs in a logical, non-redundant way should be involved.

Author Response

"Please see the attachment"

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This review examines the crucial role of RNA modifications in eukaryotic gene expression, specifically how they regulate processes during early adenovirus infection and influence cell fate, highlighting the complex transcription initiation sites of adenoviral oncogenes. The authors detail the crucial reliance of early adenovirus mRNA on alternative splicing, which is essential for viral replication, and explore its potential contribution to carcinogenesis, drawing parallels with the abnormal alternative splicing observed in cellular oncogenesis. This manuscript is acceptably written; however, it requires a comprehensive review to address the following considerations.

• Major issues
  1. Clarification on Cycloheximide (CHX) Treatment: To ensure readers from diverse backgrounds fully grasp the biological implications of the experiment, it would be beneficial to explicitly state, even briefly, that CHX is a protein synthesis inhibitor. This crucial context will help readers understand why the accumulation of early mRNA might differ in its presence or absence, thereby enriching the biological background.
  2. Precision in Terminology for "charge size": The phrase "charge size" in the context of column chromatography can be ambiguous. Consider replacing it with more precise scientific terminology. Depending on the exact mechanism of separation, more suitable phrases might include "charge properties and molecular size," "charge-to-mass ratio," or "electrophoretic mobility" to enhance clarity and scientific accuracy for a broader academic audience.
  3. Distinction and Consistency of RNA Terminology: Clarification is needed regarding the terminology used for Poly(A) RNA and Poly(A) mRNA. Explain the intended distinction between these terms, especially how they relate to each other within the context of your discussion. Additionally, ensure consistent usage of "RNA" versus "mRNA" throughout these lines (e.g., "Poly(A) RNA" vs. "Poly(A) mRNA," and "Ad2 pre-mRNA" vs. "Ad2 early mRNA"). For instance, in lines 166 and 167, specifying whether it refers to generic RNA or messenger RNA would improve precision.
  4. Elaboration on VA RNA's Mechanism via PKR Inhibition: While the manuscript notes that "VA RNA promotes adenovirus replication within cells," it would significantly enhance the discussion to elaborate on the well-established mechanism of its action. One of the most classic and widely recognized functions of VA RNA is its ability to inhibit the activity of the host cell's Protein Kinase R (PKR). Incorporate details from relevant literature to explain how VA RNA counteracts the host's antiviral response, providing crucial mechanistic depth.
  5. Detailed Mechanism of Viral Replication Inhibition by mivaRNA/RISC: The text states that "small RNAs derived from VA RNAII are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC)." Provide further detail on how these RISC-incorporated RNA molecules specifically exert their function of "inhibiting viral replication." Expanding on whether this involves direct mRNA degradation, translational repression, or other regulatory pathways, supported by literature, would greatly enhance the reader's understanding of their inhibitory mechanisms.
  6. Broader Context for ncRNAs and Aberrant Splicing in Viral Replication: The manuscript effectively introduces the impact of ncRNAs on pre-mRNA splicing, citing the case of aberrant splicing in Ad40. To broaden the context, elaborate on why this aberrant splicing is crucial for viral replication. Furthermore, discuss whether similar phenomena—where viruses exploit or induce aberrant splicing patterns facilitated by host factors (like U-snRNA)—are observed in other viral systems, which would provide a more comprehensive view of this viral strategy.

                                                                                                

• Minor issues
  1. The last sentence in the abstract should be checked with “adenovirus replication.”, not “adenovirus viral replication.”
  2. Ensure that all reference numbers are consistently placed at the end of the sentence or clause to which they refer, and preferably before the final punctuation mark.
  3. Carefully review the manuscript for any inconsistent spacing or line breaks that might disrupt readability.
  4. Amend "Ad2" to "HAdV-C2" for accurate and standardized virus nomenclature.
  5. Verify and correct the formatting of superscripts, particularly for terms like "Poly(A)+" or "Poly(A)-" to ensure proper scientific representation.
  6. Maintain consistent capitalization for "PolyA" or "polyA" throughout the entire manuscript. For example, choose either "Poly(A)" or "poly(A)" and apply it uniformly.
  7. Change the colon (":") to a period (".") to ensure grammatical correctness and sentence structure.
  8. Please rephrase it to improve clarity, conciseness, and ease of comprehension. Consider breaking it into shorter sentences or restructuring the clauses.
  9. Introduce the abbreviation "IFN" for "interferon" at its first mention: "interferon (IFN)."
  10. Correct "RIG-1" to "RIG-I" for the proper nomenclature of the gene/protein.
  11. Verify the consistent use of virus names and their corresponding abbreviations throughout the manuscript. Ensure that full names are given at first mention, followed by their abbreviations where appropriate.
  12. Correct the merged word "MLP-TSS-sRNAinhibit" to "MLP-TSS-sRNA inhibit" for proper spacing.
  13. Thoroughly review the content within lines 407 to 411 for clarity, accuracy, and flow, making any necessary revisions.

Comments on the Quality of English Language

Numerous errors are apparent throughout the manuscript, necessitating a thorough and comprehensive check.

Author Response

"Please see the attachment" 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

My comments:

L53-54: sentence "CHX blocks the translocation reaction of 53
transfer RNA on ribosome during protein synthesis in eucaryotic cells." is misplaced in the text. It has to be after sentence "Thomas and Green demonstrated that adding the protein synthesis inhibitor cycloheximide (CHX) to adenovirus-infected cells distinctly separates the synthesis of viral mRNA into the early and late phases of infection [12]. ". 

L56: mouse polyoma PyVs, use acronyme MPyV.

L80-82: totally unclear meaning of sentences, remove or rephrase

L85: "clearly" is overstated, as it is not a clear review

L352: wrong! mivaRNAS have never been shown to inhibit virus infection! 

L354-357: The reference is lacking for this statement.

L529-533: Unclear paragraph. Delete.

L534-539: This paragraph is badly placed in the text. Totally unclear what the authors want to say. Write it as a separate paragraph about MLTU alternative splicing.

L583: "occurs" is a too strong a statement

L650: "We could have made another proposal in this review. " This is very unclear as it is impossible to understand what is the "another proposal" mentioned by the authors.

L653-655: irrelevant, remove.

 

 

 

 

 

 

 

 

Comments on the Quality of English Language

Has to be improved, and a REAL English-speaker and someone who can combine sentences and paragraphs in a logical, non-redundant way should be involved.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Dear Authors,

We are pleased to acknowledge that the revised manuscript demonstrates significant improvements and enhanced clarity.

Best regards,

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

L345-347: The sentence “It has become clear that small non-coding RNA plays a crucial role in the replication of human adenovirus within human-derived cells. These are VA RNA, mivaRNA, and MLP-TSS-sRNA.” Has several problems. 1) small non-coding RNA has to be defined here as sncRNA as this acronyme is used later in the text, 2) the same applies to human adenovirus, why not to use the acronyme here, 3) the statement is wrong as mivaRNAs, as I pointed out in my previous review, ARE NOT crucial for HAdV replication. This has been shown by Kamel et al., 2013. So, these 2 sentences have to be rephrased to make them correct and not to provide a wrong impression to the reader.

L351-352: Do not use VA1 and VA2, the gene names are still VA RNAI and VA RNAII. Also the correct way of writing is VA-RNA and not VA RNA!

L378: VAII use VA-RNAII instead

L393:There is no such thing as “tumor genes”. There are oncogenes, tumor suppressor genes, or cancer-associated genes. Rephrase it.

L534-539: This part is very wrong! First of all, the authors should also include the review papers by Akusjärvi & Stevenin (2003) “Remodeling of the host cell RNA splicing machinery during adenovirus infection” Current Topics in Microbiology and Immunology, Törmänen Persson & Akusjärvi (2005) “Adenovirus RNA processing” Virus Research, as the general reviews about HAdV splicing. Second, the paper by Muhelman is not the key paper; use the data from Kanopka et al, 1996, 1998 instead, as the mechanistic studies explaining the details of the HAdV splicing. Third, for the information to the authors, the 3VDE is cis-acting RNA splicing enhancer (i.e., RNA seqiunce) and not a protein factor. 

L623-625: “. This conclusion is supported not only by studies of the 5'-terminal cap structure of mRNA but also by numerous other studies as described in the summary of each paper.”. This is an unclear sentence; does it mean that the reader should read the “summary” (do the authors mean abstract?) of every cited paper? We do it anyway, right?

L634-636: The statement “In this review article, we proposed a possible relationship between the emergence of diverse TSSs for adenovirus early mRNA and the diversity of the E1a protein's transactivation activity.” is not correct, as the review does not discuss E1A transactivating functions. There are several good reviews about E1A functions by Joe S. Mymryk’s lab. I strongly recommend removing this sentence as it is not correct regarding the review context.

L634-636: Remove this part as it is too detailed and does not fit with the conclusion section that the authors try to put forward.

Rebuttal letter: I do not agree with the author's statement “….One of the authors of this paper is among those who advanced the research and development of modification molecules for both of these RNAs. From that perspective, we believe this paper has provided clear observations regarding past research findings, at least concerning viral RNA-derived modification molecules and mRNA transcription start sites.” The fact that a scientist has been involved in performing the key experiments and driving the research field forward does not mean that the standards of the review writing should not be followed. I am not challenging the original studies and their impact, but rather the way this review has been written. It would be just sad if the elegant studies by Dr. Hashimoto were not put into an HAdV biology context in a readable, understandable way to attract readers.

 

Comments on the Quality of English Language

Can be improved.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I do not want to be mean, but honestly, this is the most unreadable review I have ever read! There are so many concerns regarding it so that I stopped in the middle of the manuscript as I did not want to waste my time. Although I think that the review topic is important and needed, the way it has been written, both fact-based and grammar-based, it is just unacceptabel! In my mind the review has to be something that is easy to read, makes you think after, generates you new ideas and updates your knowledge. It should be readable both at the undergraduate as well as at a Professor levels. Furthermore, a good review should contain at least one figure to make it appealing to read and to memorize the topic. Unfortunatelly all these charateriztics are missing in this review.

Here are some of my comments before I gave it up:

L25: use "constitutive splicing" instead of "splicing"

L28-29: sentences not needed as they do not contribute to the abstract understanding

L30-31: sentences not needed as they do not contribute to the abstract understanding

L39-40: If other viruses have acronyms, then also cytoplasmic polyhedrosis virus and reovirus should have acronyms. The authors should also be consistent with the acronyms throughout the manuscript. Why not to use HPV instead of human papillomavirus

L44: vaccinia virus use acronyme

L45: What are caps? In this context, it sounds like slang. I have to be defined.

L46: Since these studies were done with human adenoviruses, it has to be defined

L51: Cyclohexamide has an acronyme CHX not CH!

L58: where it is discussed? The authors write multiple times "this will be discussed....", however it remains unclear what and where will be discussed. The authors use word "discuss" 16 x in the manuscript, which makes it difficult to understand what they at all want to discuss....Avoid using it soooo many times.

L59: sentence not needed as it is repetition

L71-73: These sentences do not say too much, remove

L87-90: Rephrase, avoid "discuss"

L94-95: Rephrase or delete. The authors should use less the word "focus" as there is no real focus in the manuscript

L105: "seven types of virus-infected cells"? Well, for sure more than 7 different cell lines have been used for these studies throughout the years...

L107/L140: Rephrase, avoid "discuss"

L115-120: Not needed, repetion of the text

L124-164: Very difficult to understand this paragraph, remove or rephrase it to have a clear text and point 

L209-213: Totally irrelevant text! Remove as it does not fit into the context

L222: Define "Writer-Reader-Eraser" for a reader

L232: 2 out of 3 references are wrong, keep only Sommer at al.

L238-242: The text is redundant, and has to be shortened

L243-244: Why this statement? It is general knowledge.

L339-346: This part has to be rewritten! VA-RNAs are considered as small noncoding RNAs and they can be processed into mivaRNAs.This part has not been explained at all, so the reader is really puzzled about the present text. Particularly, L341-342 is a nonsensical sentence that does not make any sense.

L350-353: Wrong statements! It is ridiculous to base it on a review article without checking the details! HAdV2/5 have never been shown to express the TSS-RNAs, it is only shown and proved in the case of the HAdV-37! The authors should really read the previous review not make wrong statements!

L362-363: unclear whose work the authors refer to.

L376: again unclear who has made this statement and what is U-snRNA, which pops up out of the "blue"

L390-417: This paragraph has to be updated! There are several elegant studies with NGS data where the authors have mapped in detail the TSS, splice sites, TTS, and so on. To write this paragraph only on data from "old good days" is just a kind of statement of ignoring the present NGS data-sets.

At this point I gave up....

 

 

 

Comments on the Quality of English Language

The language and writing styles are just not OK! Almost impossible to understand what the authors mean and what the red line ("take home message") is in some parts of the review. The review has to be rewritten, as in the present form, it can not be accepted at all. There is a need for a professional writer, as obviously AI has not helped the authors at all. Or is it the opposite? AI has generated a big, messy manuscript?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Hashimoto et al. intends to review the transcription initiation process in viruses and the mRNA splicing.of viral RNA. 

Although the issues intended to be analyzed are significant, it is not possible to understand the content of the overall manuscript. This is because the manuscript needs extensive language editing. It is not possible to go through the text and understand its main ideas. I believe it cannot be published in the present form.  

Comments on the Quality of English Language

It needs extensive English editing to make the manuscript content clear.  

Author Response

Please see the attachment.

Author Response File: Author Response.pdf