LncRNA RORB-IT1 Encoding a Micropeptide Regulates Progesterone Synthesis, Proliferation and Apoptosis in Chicken Granulosa Cells

Comments 1: Although the study clearly demonstrates functional effects of RORB-IT1 and RORB-34aa on progesterone synthesis, proliferation, and apoptosis, the downstream molecular mechanisms remain unclear. The authors are encouraged to explore or at least discuss potential signaling pathways (e.g., cAMP/PKA, MAPK, PI3K-AKT, mitochondrial apoptosis pathways) through which RORB-IT1 and RORB-34aa exert their effects.

Response 1: Thank you for pointing this out. We agree with this comment. We have expanded the Discussion section to include a discussion on the potential molecular mechanisms and signaling pathways involved. Please refer to the last paragraph (lines 470–481) on page 13 of the revised manuscript.

Comments 2: The manuscript reports that RORB-IT1 inhibits apoptosis, whereas its encoded micropeptide RORB-34aa promotes apoptosis. This paradoxical effect is intriguing but insufficiently explained. The authors should expand the Discussion to propose mechanistic hypotheses explaining how RNA-level and peptide-level functions diverge and how cellular balance between the two is regulated.

Response 2: Thank you for your valuable suggestion. We have added a hypothesized explanation for the "RNA-peptide functional separation" mechanism in the Discussion section. We propose that RORB-IT1 may inhibit apoptosis through its function as an RNA scaffold, while RORB-34aa may promote apoptosis by binding with regulators interfering with signaling cascades such as MAPK or PI3K-AKT. The two exert their effects at different hierarchical levels of the signaling network, resulting in functional antagonism. Please refer to the last paragraph (lines 470–481) on page 13 of the revised manuscript for these additions.

Comments 3: Detection of RORB-34aa relies primarily on overexpression systems with EGFP tagging. The authors should clarify whether endogenous RORB-34aa protein expression is detectable (e.g., by mass spectrometry, peptide-specific antibody, or ribosome profiling). If not feasible, this limitation should be explicitly acknowledged.

Response 3: Thank you for the valuable suggestion. Actually, we have attempted to synthesize specific antibodies to validate the presence of the endogenous RORB-34aa peptide. However, the synthesized antibodies did not perform well, and we were unable to confirm the presence of the peptide through immunodetection. The suggestion to use mass spectrometry or ribosome profiling for endogenous peptide identification is highly constructive, and we plan to incorporate this method in future studies to confirm the presence of RORB-34aa in chicken granulosa cells. We have explicitly acknowledged this limitation in the Discussion section. Please refer to lines 502–512 on page 14 for details.

Comments 4: Most experiments are reported with n = 3. While common, this relatively small sample size may limit statistical robustness. The authors should clarify whether these represent independent biological replicates from different animals and consider discussing statistical power limitations.

Response 4: Thank you for the valuable suggestion. In this study, each experiment was independently repeated 3 times, with three hens used per experiment. We recognize the importance of increasing biological replicates to enhance statistical power and reliability. We have added a clarification in the statistical analysis section (Section 2.1) stating that all experiments were conducted using three independent biological replicates. Please refer to lines 100–102 on page 3.

Comments 5: The concentrations of FSH, E2, and P4 used in vitro should be better justified with references or physiological relevance to in vivo follicular environments.

Response 5: Thank you for your suggestion. We have supplemented the rationale for the selected hormone concentrations in the hormone treatment section (Section 2.2), citing relevant references. Please refer to lines 128–130 on page 3 for details.

Comments 6: Several sentences require minor grammatical corrections and stylistic polishing. For example: “might be relating with chicken egg production” → “might be related to chicken egg production”， “this classic signaling cascades” → “these classic signaling cascades”.

Response 6: Thank you for your suggestion. We have corrected grammatical errors and improved inaccurate expressions throughout the entire manuscript. Please refer to the revised manuscript for the complete updates.

Comments 7: Some figures (e.g., Figures 6 and 7) contain many panels and would benefit from clearer labeling and slightly larger font sizes. Scale bars should be consistently indicated and described in all figure legends.

Response 7: Thank you for your suggestion. We have improved Figures 6 and 7, increasing the font size and improving the clarity of labels. Furthermore, we have ensured that scale bars are clearly indicated and described in all figure legends. Please refer to the updated figures in the revised manuscript.

Comments 8: In Figure 5E, Bcl2 expression is reported to be reduced after RORB-IT1 overexpression, which appears contradictory to reduced apoptosis. The authors should clarify or discuss this observation.

Response 8: Thank you for your suggestion. We have supplemented the explanation in both the Results section corresponding to Figure 5 and the Discussion section: For details, please refer to Section 3.5, lines 355–360 on page 10, and lines 482–494 on page 14 of the Discussion section.

Comments 9: Some background references on lncRNA-encoded micropeptides could be updated with more recent studies (2022–2025) to strengthen the introduction and discussion.

Response 9: Thank you for your suggestion. We have updated the relevant references in both the Introduction and Discussion sections, citing recent studies (2023–2025) on lncRNA-encoded micropeptides, such as Guo et al. 2023, Yu et al. 2024, Zhang et al. 2025, among others. For details, please refer to the Discussion section and the references.

Comments 1: In this study, EGFP fusion proteins were used to evaluate the function of the 34-amino-acid micropeptide (RORB-34aa) derived from RORB-IT1. However, it is not clearly distinguished whether the observed biological effects are attributable to the 34-residue peptide itself or to properties of the fusion protein. Although the use of initiation codon mutants to demonstrate translation dependency is appropriate, the current data appear insufficient to conclude that RORB-34aa alone constitutes a fully functional unit under endogenous conditions. The authors are therefore encouraged to adopt a more restrained interpretation of the micropeptide’s function or to more explicitly state that the functional analyses were performed using fusion proteins.

Response 1: Thank you very much for pointing this out. We apologize for the errors in describing the plasmid construction and in drawing Figure 6B. Actually, we only used the EGFP fusion expression plasmid to detect the translation and location of micropeptide RORB-34aa because the RORB-34aa is too small. We inserted the RORB-34aa coding sequence with or without ATG mutation into pcDNA3.1(+) overexpression vector to evaluate the effects of RORB-34aa on the proliferation, apoptosis and progesterone synthesis in granulosa cells. Certainly, maybe we can synthesize the micropeptide RORB-34aa in vitro and add to the culture medium to detect the function of it in future. We have made the corresponding corrections in the Material and Methods (Page 4, Lines 157-160) and Figure 6B.

Comments 2: Although the authors demonstrate the theoretical feasibility of RORB-34aa translation, they do not provide direct evidence for its actual translation and presence under endogenous conditions, such as detection using a specific antibody, or mass spectrometry-based approaches. Consequently, the physiological existence of this peptide remains unresolved. It would be important to clearly acknowledge this limitation of the present study—namely, the lack of detection of endogenous RORB-34aa—and to highlight it as a key subject for future investigation.

Response 2: Thank you for the valuable suggestion. We absolutely agree with this. Although we have attempted to synthesize specific antibodies to validate the presence of the endogenous RORB-34aa peptide, the synthesized antibodies did not perform well. We will use mass spectrometry or ribosome profiling to confirm the presence of RORB-34aa in chicken granulosa cells in future studies. We have revised the Discussion section to more clearly acknowledge the lack of endogenous peptide detection as a key limitation of the present study and to highlight this as an important direction for future investigation. The following sentences have been revised in the Discussion section (Page 14, Lines 502-507).

Comments 3: The manuscript reports the intriguing observation that the RORB-IT1 RNA itself promotes granulosa cell survival and progesterone synthesis, whereas RORB-34aa promotes cell proliferation while simultaneously inducing apoptosis. However, it remains unclear which of these effects predominates under endogenous conditions, or how their temporal and quantitative relationship is regulated in vivo. As a result, although the concept of "dual regulation" is proposed, the relative physiological significance of each component has not yet been fully resolved.

Response 3: Thank you for pointing this out. We agree with this comment. We are also interested in the observation that RORB-IT1 RNA and RORB-34aa exhibited paradoxical effects on apoptosis. We speculated that the RORB‑IT1 RNA may function as a scaffold or ceRNA, while the RORB‑34aa peptide may bind with critical regulators within pathways such as MAPK or PI3K/AKT, leading to opposite apoptotic outcomes. We will integrate transcriptomics, phosphoproteomics, IP-MASS, and others to decipher how this intramolecular feedback loop is orchestrated in future studies. Moreover, in vivo experiments need to be performed to determine the relative physiological significance of each component. We have revised the Discussion section. Please see Page 14, Lines 470-481.

Comments 1: The highlights and novelty of the research are well presented at the beginning of the manuscript, although I suggest that the last sentence from the Conclusions nicely can fit at the end of Introduction (after the aim).

Response 1: Thank you for this insightful suggestion. We have moved this sentence to the end of the Introduction section to emphasize the significance potential of our study (Page 2, the last Paragraph, Lines 90–93).

Comments 2: Please develop a flowchart for the design of the M+M.

Response 2: We appreciate this suggestion. A schematic flowchart summarizing the overall experimental design has now been prepared and included as Supplementary Figure S2.

Comments 3: Please add the year at the Ethical Committee permission.

Response 3: We apologize for this omission. The year of ethical approval has now been added to the Institutional Review Board Statement (Page 3, Paragraph 3, Lines 113–115).

Comment 4: Please indicate the study duration (from–to).

Response 4: Thanks for your suggestion. Our study duration is from 2023 to 2025. We performed this study after all experimental procedures were approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University (code SDAUA-2022-36). But we feel that it is not suitable to add them in the manuscript.

Comment 5: I would like kindly ask the authors to add references at almost all methodology subsections starting from 2.2 until 2.13.

Response 5: Thank you for this important methodological suggestion. We have now added appropriate references to nearly all subsections from 2.2 to 2.13, citing established protocols and previous studies, including our recent work [23]. For common commercial kits, we have cited the manufacturer’s instructions or standard protocols. Sections 2.2–2.13, pages 3–6; new references [19–24] added.

Comment 6: Please include for the micrographs of Figs. 4C, E; 6A; 7B the used methods and shortly indicate what is visible in each of them.

Response 6: Thanks for pointing this out. We agree that micrograph illustrations should be self-explanatory. The figure legends for Figs. 4C, 4E, 6A, and 7B have now been revised to clearly state the method used (e.g., EdU assay, EGFP fluorescence) and briefly describe the visible signals (e.g., EdU-positive nuclei in red, total nuclei in blue, EGFP-tagged peptide in green). Please refer to the revised manuscript for the complete updates.

Comment 7: Please add the Limitations at the end of the Discussion chapter.

Response 7: Thanks for pointing this out. We have now added the limitations in the Discussion section, addressing the following points: (1) lack of direct endogenous peptide detection; (2) in vitro model limitations; (3) the regulation mechanisms need to investigation. Please see Page 14, Paragraph 4, Lines 502–512.

Comment 8: I would prefer to read also something about the influence of confounding factors on the expression of RORB-IT1.

Response 8: Thanks for your valuable suggestion. A new paragraph discussing potential confounding factors—including genetic background, age, nutritional status, stress, photoperiod, circadian rhythms, and epigenetic modifications—has been added to the Discussion section, supported by relevant references [36–39] (Page 13, Paragraph 1, Lines 434-437).

Comment 9: Conclusions. Please remove the extra words and the last sentence.

Response 9: Thanks for pointing this out. We have revised according to your suggestion. Please see Page 2, the last Paragraph, Lines 90–93.