PONE-D-25-03086PRAME-AS lncRNA is regulated by MZF1 and is involved in the expression of PRAME transcripts and cell stemnessPLOS ONE

Dear Dr. Dehghani,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  This study demonstrates that a lncRNA (PRAME-AS) can regulate its homologous coding gene PRAME. The authors identify MZF1 and hypermethylation of the upstream regulatory region as regulators of this lncRNA's transcription, and ultimately reveal that the PRAME promoter functions unidirectionally in regulating PRAME. While this discovery is novel, the main findings require further experimental validation to strengthen their impact.

Main concerns:

1. The title emphasizes the lncRNA and expression of PRAME, but the narrative lacks cohesion: The authors first identify the lncRNA, after knock out, the PRME expression is reduced, how does LncRNA regulate PRME? without explore further, the author turned to check cell proliferation, migration, viability, and anchorage-independent growth, then they turn to the explore its upstream regulatory factors (MZF1 and hypermethylation), and later shift to analyzing bidirectional promoter activity, Please ensure that all sections are connected naturally and smoothly.

2. The abstract uses non-definitive phrases ("lncRNA MIGHT regulate PRAME", "MZF1 MAY affect both genes"). If the results are reliable, replace tentative terms ("might/may") with stronger language (e.g., "demonstrates" or "mediates") to enhance scientific rigor.

3. PRAME-AS lncRNA transcripts show no significant differential expression between normal and tumor tissues. This raises concerns about its functional role in tumor progression. what drives authors continues to explore this LncRNA in this situation?

4. The study attributes PRAME-AS-mediated phenotypes (proliferation, migration, etc.) to lncRNA activity, but it remains unclear whether these effects are PRAME-dependent or not. To confirm that the observed phenotypes are specifically regulated by PRAME-AS (and not solely by PRAME), include PRAME knockdown/knockout controls in functional assays.

Minor points

1. Figure 1: Include PRAME protein expression to complement transcript-level findings. This could be added as supplementary data.

2. Figure 2G Provide PRAME protein expression results following PRAME-AS knockout to validate transcriptional regulation at the protein level.

3. Figure 4 Include Western blot data to confirm successful MZF1 overexpression.

4. Figure 6C Clearly the cell number of AS direction is much less than the other two.

Reviewer #2:  In this paper, titled “PRAME-AS lncRNA is regulated by MZF1 and is involved in the expression of PRAME transcripts and cell stemness”, the authors have undertaken a characterization of a previously unstudied lncRNA originating antisense from tumor antigen PRAME (PRAME-AS). Regulation of PRAME-AS has been analyzed via interrogation of known expression datasets, transcription factor overexpression, the efficacy of its promoter region and the extent of CpG methylation surrounding it. The function of PRAME-AS was assessed via Cas9-mediated ablation with downstream impacts on PRAME expression, proliferation and cell migration.

It is remarkable that for a gene with long known implications in tumorigenesis that the antisense lncRNA expressed at this locus has so far gone uncharacterized and so the authors should be commended for tackling this as a research topic. The method of PRAME-AS KO so as not to alter promoter activity of PRAME is elegant, however there are several concerns regarding the veracity of the conclusions until further clarity is reached.

Major Comments:

Figure 2

1) What is the expression level of PRAME-AS in HEK293T cells and how does it compare to PRAME expression approximately? Low expression levels is not necessarily an impediment to study as lncRNAs are commonly lowly expressed, but it should be made clear relative to housekeeping gene.

2) What is the extent of PRAME-AS decrease after integration of the donor fragment? Preferably shown with primers both upstream (Exon 2) and downstream (Exon 3) of the integrant.

3) There is concern that inserting a construct inversely to PRAME-AS but on the same strand and upstream as PRAME may lead to interference with PRAME transcription if there is transcription readthrough of the construct.

a) Does the sequence have (a) polyadenylation signal(s) to ensure transcription termination of the integrant?

b) Can it be shown that there is no transcription readthrough downstream of the 3’TR region?

4) Figure 2F: The presence of the Puro-IRES-GFP integrant is demonstrated, however it isn’t clear or explicitly stated whether this is integrated into or one both alleles. Can this be shown or clarified?

Figure 3

1) It would be ideal to perform a rescue experiment of overexpressing PRAME in the PRAME-AS KO cells to determine whether those phenotypes are due to the ~34% loss of PRAME.

Figure 4

1) Related to Figure 2, the fold change of PRAME and PRAME-AS can be seen but how do they compare to each other? It would be very interesting to see similar fold increases when overexpressing MZF1 if there were a large basal difference between PRAME and PRAME-AS.

Figure 5

1) If PRAME-AS expression levels are already low, and methylation levels high, then any dCas9 methylation will likely not alter PRAME-AS expression levels further. Can the level of methylation of each of the CpGs analyzed with bisulfite sequencing be quantified?

2) The images look as though transfection efficiency is not 100%, which likely dampens the impact on expression differences between negative control and dCas9-DNMT1 plasmids, which may be especially relevant if the levels of PRAME-AS are already low. Can the GFP+ cells be enriched via cell sorting to parse this out, or can the authors speak to this possibility?

3) The title of the figure states that hypermethylation diminishes PRAME-AS transcripts yet the quantification in Figure 5D is not significant, this should be corrected or clarified.

Figure 6

Here the authors are attempting to determine whether PRAME-AS is the result of a bidirectional promoter (from PRAME). Here, the luciferase results from Schenk et al (2007) are used as a marker for determining where the maximum promoter activity for PRAME is located. In that paper, PRAME is shown to have two transcription start sites (TSS) annotated, in which the downstream one has stronger activity, and may be the source of the bidirectionally of PRAME-AS, thus the overlap of the 5’ exon is due to annotation of the upstream PRAME TSS with the antisense transcription of PRAME-AS resulting from the downstream PRAME TSS. Can the authors speak to this possibility?

Bidirectional transcription from protein-coding genes often contain promoter proximal polyadenylation signals in the antisense direction (in this case PRAME-AS). Can the authors confirm whether the “PRAME-AS direction” construct contains any AATAAA/ATTAAA signals that may prematurely terminate transcription upstream of GFP?

A much more convincing demonstration of bidirectional promoter activity would be to only include the CpG island region as shown in Figure 5 (perhaps overlapping PRAME promoter as well), if possible even including +/- in vitro construct methylation by SssI as shown in Shenk et al. Alternatively, can it be explained in more detail as to how this region is not "bidirectional" based on the tests that have already been completed?

Minor Comments

1) Figure 1: Font of y-axis is very small, should be enlarged to scale with the rest of the graph

2) Figure 1: It is very interesting that PRAME-AS appears to be completely silenced in testicular germ cell tumors. What is the p-value of the comparison between testicular germ-cell tumors and normal testicular cells

3) It is fine to reference a prior paper for details on the methods regarding TransCRISTI, however the methods section of this publication should still have a section dedicated to it, even if it is only to refer to the prior paper.

4) Figure 5: Can it be labeled more explicitly which sgRNA corresponds to which CpG island?

2) Figure 4: What happens to PRAME and PRAME-AS expression if MZF1 is upregulated in PRAME-AS KO cells? Is there a link between MZF1 recruitment and PRAME-AS influence on PRAME?

5) Figure 5: Could the authors show that reducing DNA methylation at the PRAME-AS ‘promoter’ leads to increased PRAME-AS expression i.e through dCas9-Tet1?

Reviewer #3:  Overview

In this study, the authors investigate the effects of long non-coding RNA (LncRNA) PRAME-AS gene on the expression of PRAME, cell proliferation, migration, cell viability and anchorage-independent growth with results showing a decrease in all of these functions. Upregulation of PRAME-AS via MZF1 overexpression and hypermethylation lead to an increase in the above-mentioned characteristics.

Overall, this study was written clearly and the work was well performed.

Comments

Major comments:

1. A full description of the TCGA and GTEx datasets for bioinformatics analyses should be included in M&M.

2. Some of the results sections describe details of the technique used more than an elaborate description of the actual results and their significance.

3. More focus on the significance of the findings is needed in the discussion.

Minor comments:

Tables 1 & 2 can be added to supplementary information.

Reviewer #4:  Hosseininia and Dehghani investigated the role of the antisense lncRNA of the PRAME gene (PRAME-AS) in PRAME regulation and cell phenotypes. The authors revealed that i) PRAME-AS knockout decreased PRAME mRNA levels by approximately 34%, ii) cell migration, viability, and stemness were decreased in PRAME-AS knockout cells compared to control cells, iii) MZF1 increased the expression of PRAME-AS and PRAME transcripts, and iv) DNA hypermethylation of PRAME-AS upstream downregulated the expression of PRAME-AS and PRAME transcripts.

The authors concluded that PRAME-AS regulates PRAME at the transcriptional level. However, the data provided are insufficient to support this conclusion.

Major Comments:

1. Although the authors used HEK293 cells to investigate the effects of PRAME-AS on cell stemness, these cells were not optimal for the stemness assay due to their insufficient colony size for demonstrating differences. Furthermore, the function of PRAME is well-established in cancer cells. The authors should use at least one cancer cell line in addition to HEK293 cells. The utilization of more than two cell lines would be preferable for generalizing their findings.

2. The authors should investigate the effect of PRAME-AS on cell growth. The number of control and PRAME-AS knockout cells should be counted from the initial day of culture until day 3 or 5.

3. Despite being statistically significant, the observed reduction in PRAME mRNA levels and the diminished stemness and migratory capabilities resulting from PRAME-AS knockout appear to be minimal. These subtle changes may not result in substantial biological effects. The authors should provide an explanation for the modest decrease in PRAME mRNA and elucidate why there are only slight differences between the wild-type cells and those with PRAME-AS knockout in both the soft agar and scratch assays (Figures 2G, 3C, and 3D).

4. Although the authors avoided CpG island on the primers for bisulfite sequencing (page 10, line 162), DNA methylation of CpG island is crucial for the regulation of gene expression. Primers should be designed to analyze CpG methylation on CpG islands.

5. Figure 6A should be carefully edited for the following reasons:

a) Although the authors stated that H3K27ac data of the PRAME promoter was from UCSC Genome Browser (page 25, line 495), the observed H3K27ac peak differed from the current information available on the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr22%3A22556888%2D22562353&hgsid=2476779099_S0v0GSEFx5vKwQS2lhUHcGN4aBHD).

b) The positions of CpG island are based on a previous study (Schenk T et al, ref#22). However, this is the predicted position, and the current official annotation of CpG island locations can be found in the UCSC Genome Browser.

c) It is unclear how to generate the continuous peak of PRAME promoter activity from the intermittent data obtained through the luciferase assay.

6. The authors should carefully edit the entire manuscript:

a) The Discussion section needs revision to eliminate redundancies and excessive length; for examples, the detailed descriptions of other antisense lncRNA are not necessary (page 27, lines 534 – 548), the detailed information of MZF1 (page 29, lines 574 – 595) should be shorten because the regulation of PRAME by MZF1 has been reported in previous papers (Lee et al, ref#23 etc.), repeated description of the results are not necessary (page 30, lines 612 – 616).

b) “CPG” should be changed to “CpG” throughout the manuscript.

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Reviewer #1: No

Reviewer #2: Yes:  Michael Robert Murphy

Reviewer #3: No

Reviewer #4: No

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PONE-D-25-03086R1PRAME-AS lncRNA, regulated by MZF1, modulates PRAME expression and cell stemnessPLOS ONE

Dear Dr. Dehghani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript was reviewed by three referees who had reviewed your original one. Basically, they are satisfied with your revision, but one of them suggested some minor issues to be resolved. Please revise it according to their suggestions.

Please submit your revised manuscript by Sep 03 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Minor comments for Hosseininia & Dehghani:

1. Figure 2A spelling: “PRAM-AS”

2. Wording regarding unidirectionality – PRAME-AS transcription appears to be dependent on PRAME transcription. I don’t think it is correct to say that “PRAME promoter operates unidirectionally, regulating only PRAME transcription”. PRAME promoter clearly regulates PRAME-AS as well through bidirectional transcription. Perhaps more accurate to say that PRAME-AS strand promoter region is insufficient for transcription (based on reporter expression) without PRAME transcription (the PRAME strand) to drive its expression.

3. In the discussion it is mentioned that a possible regulatory mechanism may involve direct interactions between PRAME and PRAME-AS via RNA-RNA duplex. A BLAST search of spliced PRAME-AS compared to PRAME entire transcript (exon and intron) seems to indicate that ~100nt of PRAME-AS exon 3 has complementarity with PRAME intron. If RNA-RNA duplex is mentioned as a possibility, then evidence or lack of evidence of this through sequence search should be done.

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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Reviewer #2: Yes:  Michael R. Murphy

Reviewer #3: No

Reviewer #4: No

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PRAME-AS lncRNA, regulated by MZF1, modulates PRAME expression and cell stemness

PONE-D-25-03086R2

Dear Dr. Dehghani,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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Reviewer #2: Yes:  Michael R. Murphy

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