NORFA, long intergenic noncoding RNA, maintains sow fertility by inhibiting granulosa cell death

Long intergenic non-coding RNAs (lincRNAs) have been proved to be involved in regulating female reproduction. However, to what extent lincRNAs are involved in ovarian functions and fertility is incompletely understood. Here we show that a lincRNA, NORFA is involved in granulosa cell apoptosis, follicular atresia and sow fertility. We found that NORFA was down-regulated during follicular atresia, and inhibited granulosa cell apoptosis. NORFA directly interacted with miR-126 and thereby preventing it from binding to TGFBR2 3′-UTR. miR-126 enhanced granulosa cell apoptosis by attenuating NORFA-induced TGF-β signaling pathway. Importantly, a breed-specific 19-bp duplication was detected in NORFA promoter, which proved association with sow fertility through enhancing transcription activity of NORFA by recruiting transcription factor NFIX. In summary, our findings identified a candidate lincRNA for sow prolificacy, and provided insights into the mechanism of follicular atresia and female fertility.

and the conclusion is drawn from the purpose of the study and the results obtained from the experiments. Appropriate references have been cited when necessary in most cases for a comprehensive understanding of the study. The manuscript is written in standard English. However, the following revisions need to be made.
Specific comments Revise the following statements at abstract to make them meaningful and clear; • The first sentence in the abstract (line 8) does not express any coherent idea. It seems to suggest that "lincRNAs have been implicated in healthy and disease conditions". Restructure the statement to reflect so, if that is what the sentence seeks to suggest. • Line 15-16 (furthermore, the correlations among NORFA, miR-126 and TGFBR2 levels were validated in follicles. SUGGESTION: The correlation between NORFA, miR-126, and TGFBR2 levels in follicles was further validated) The introduction or background of the study has no heading/subheading (line 22). Cite reference for the ideas expressed in the statement at lines 35 to 37 of introduction. At results section, line 108-109, the statement is ambiguous, restructure it to indicate the expression level of the four genes were increased. The meaning of the statement at discussion (line 349-350) is not clear. Revise it to capture what you want to you want to express.
BEST WISHES.
Reviewer #3 (Remarks to the Author): In this manuscript by Du et al., authors identified that pig-specific lncRNA NORFA can regulate granulosa cell apoptosis by acting as a sponge for miR-126, and further demonstrated that NORFA/miR-126 axis plays an important role in regulating GCs apoptosis through targeting TGFBR2. In the end, authors identified a pig-specific 19-bp duplication in NORFA promoter, which could regulate NORFA transcription by altering the recruitment of NFIX to the promoter of NORFA.
Overall, this study is very interesting and authors provided comprehensive experiments. However, the results were not solid enough to support the conclusions. Below are my major comments.
1, Authors stated this lncRNA is pig-specific, but did not provide any evidence. Author mentioned the homologous sequence of this transcript was not detected in other mammals, however, RNA structure of this lncRNA could be conserved in other mammals.
2, Authors did not provide negative control for their FISH experiments. To demonstrate the specificity of FISH probe, authors could include siRNA against NORFA and compare the signal and localization in the cells.
3, The center part of this manuscript is NORFA serve as a sponge for miR-126, although authors provide multiple line evidence, it is still not convincing. However, authors should perform RNA pulldown assay in the cells, at least by overexpressing NORFA and miR-126, rather relying on in vitro RNA binding assay. More importantly, authors should mutate miR-126 binding sites in the construct of pcDNA3.1-NORFA and overexpress it in cells to see whether NORFA-mu could still be able to reduce the expression of miR-126 and other responding pathways. 4, Authors state this lncRNA has a important role for sow fertility, however, the evidence provided here is not sufficient to draw any conclusion on it.

Reviewer #1 (Remarks to the Author):
Du X et al. identified nove lincRNA, NORFA from SMAD4-silenced porcine GCs. They found that NORFA has anti-apoptotic activity in GC by functioning as a ceRNA of miR-126, which has pro-apoptotic activity in GC. They also found that the pro-apoptotic activity of miR-126 is vie the direct targeting of TGFbeta-R2. They confirm that anti-apoptotic activity of NORFA is through the inhibition of miR-126 to target TGFbeta-R2. In addition, they found that NORFA promoter has 19-bp duplication that is targeted by transcriptional activator, NFIX, which is highly expressed in healthy follicles. Based on these results, they concluded that NORFA functions as an inhibitor of granulosa cell apoptosis.
Overall, the data is interesting and well designed to draw conclusion. However, I have 3-concerns that (1) some of subtitle is not matching with the data (2) The manuscript is too lengthy. I think that The data of figure 2 support that NORFA is essential for GC apoptosis but low expression of NORFA in follicular atresia (Fig. 1g, h) does not mean it is essential in follicular atresia.
Response: Thanks, we have edited the subtitle and conclusion parts in this paragraph in the revised manuscript according to your advice. The subtitle have changed to 'NORFA is involved in GC apoptosis and follicular atresia', and conclusion have changed to 'All our data suggest that NORFA is essential for inhibiting GC apoptosis, and involved in follicular atresia of pigs.'.
Response: Based on the results of Fig. 2, we demonstrate that the normal expression of NORFA is necessary for inhibiting porcine GCs apoptosis, indicating that NORFA is an anti-apoptotic factor in porcine GCs. Besides, NORFA has been proved to be a non-coding RNA which mainly function as epigenetic factors.
In order to make it clear for readers, we have modified this sentence as 'indicating that NORFA is an anti-apoptotic factor in porcine GCs' in the revised manuscript. (4) line 137: The subtitle "in vivo" is not appropriate.
Response: Thanks. In order to avoid misleading readers, we have replaced 'in vivo' with 'in porcine ovarian follicles' in the revised manuscript. Response: Thanks. In order to get more clear bands, we have re-performed the western blotting assays by reducing the amount of total protein according to your advice. As shown in Fig. R1 as well as Fig. 6 in the revised manuscript, we demonstrate that ectopic expression of miR-126 dramatically reduces TGFBR2 protein level in porcine GCs, while the opposite result was observed after miR-126 silencing.  Response: Thanks. First, according to the results of Fig.1-8, we identified a pig-specific novel lncRNA, NORFA, and demonstrated that it inhibits porcine GCs apoptosis and is involved in follicular atresia by activating TGF-β signaling pathway through interacting with miR-126-TGFBR2 axis. This part mainly focus on investigating the functions and mechanisms of NORFA in modulating GC apoptosis. As we known, follicular atresia is a limiting factor for female infertility. Thus, we hypothesized that NORFA might participate in regulating sow fertility and begin to investigate the relationship between NORFA and sow fertility, and the results are shown in Fig. 9-10.
Based on Fig. 9-10, we identified a 19-bp breed-specific duplication in the core promoter of NORFA could affect its transcription, and found that it is involved in sow fertility. This part (Fig. 9-10) mainly focuses on investigating the important role of NORFA in sow breeding works. The combination of two parts makes this article more comprehensive and contributes to a depth understanding of the functions of NORFA and its role in regulating female reproduction traits. For these reasons, we believe that Fig. 9-10 is especially important and indispensable for this article.

Reviewer #2 (Remarks to the Author):
Comment and suggestions for authors: The manuscript "NORFA, a novel candidate lincRNA for sow fertility, inhibits granulosa cell apoptosis" describes the role of a lincRNA, NORFA in porcine granulosa cell (GC) apoptosis, follicular atresia, and sow fertility. The study sort to examine if NORFA was involved in GC apoptosis and follicular atresia and to determine the actual role it plays and its mechanism of action. The study results and discussion propose that NORFA sponges endogenous miR-126 in porcine GCs and prevent its binding to the 3' UTR of TGFBR2, releasing TGFBR2 to inhibit GC apoptosis and follicular atresia. The study also identified a 19-bp duplication in the promoter region of NORFA which is a sow prolificacy-associated variant that recruits the transcription factor NF1X to enhance NORFA transcription and regulation of GC apoptosis and follicular atresia.
The study was thoroughly conducted with adequate samples, replicates, controls.
The aim is clear and technically sound methodology was used to arrive at the conclusion. Sufficient data have been provided to support the claims of the study and the data is made available. The discussion is elaborate and the conclusion is drawn from the purpose of the study and the results obtained from the experiments.
Appropriate references have been cited when necessary in most cases for a comprehensive understanding of the study.
The manuscript is written in standard English.
However, the following revisions need to be made.

Response:
Thanks very much. According to your suggestions, we have revised our manuscript and answered the following questions point-by-point.

Specific comments
Revise the following statements at abstract to make them meaningful and clear; • The first sentence in the abstract (line 8) does not express any coherent idea. It seems to suggest that "lincRNAs have been implicated in healthy and disease conditions". Restructure the statement to reflect so, if that is what the sentence seeks to suggest.

Response:
Thanks. To make it clear for readers, we have restructured the statement as 'lincRNAs have been proved to be involved in regulating health and disease in organisms' in the revised manuscript according to your advice. Response: Thanks. To make it clear for readers, we have replaced this sentence as 'Acting as a ceRNA is the main function mode for lncRNAs containing the same miRNA response elements (MREs) with targets' in the revised manuscript.

Minor suggestions
Below are some recommended suggestions for some words or statements in the manuscript: Replace "we report a novel lincRNA" (line10) with (we report that a novel lincRNA),". Replace "Prevented" with "preventing" (line 13). "Are identified" with "have been identified" (line29-30). Put "are" between miRNAs and mainly (line 35). Replace "which" with "with" and "show" with "showing" (line 38). "To" with "and" (line 49). "It is little known about" should be replaced with "little is known of" (line 51).
Response: Thanks. We have revised these statements according to your suggestion.

At results;
Change "with highly expressed" to "which was highly expressed" (line 64).
Replace "is" with "to be" and "to locate" with "to be located" (line 67).
Put a full stop after "GCs" and begin the next sentence with a capital (line115). Put "of the" before NORFA (line 121). Change "physically" to "physical" (line 127).
Rephrase this statement at lines 137-138; "due to the lack of the characterization of the gene encoding miR-126 in pig". SUGGESTION: change "the lack of" to "the unavailability of data or information on". Change "highly" to "high" (line 140).
Response: Thanks. We have revised these statements in the discussion part according to your suggestion.
Change "presenting" to "present" (line 615). Replace "served" with "use" (line 620). Put "tissue" in front of "sample" (line 632). Overall, this study is very interesting and authors provided comprehensive experiments. However, the results were not solid enough to support the conclusions.

Response
Below are my major comments.
1, Authors stated this lncRNA is pig-specific, but did not provide any evidence. Author mentioned the homologous sequence of this transcript was not detected in other mammals, however, RNA structure of this lncRNA could be conserved in other mammals.

Response:
Thanks. According to your advice, we have analyzed the structure conservation of porcine NORFA among other mammal species with the strategy below (Fig. R3a). First, we analyzed the secondary structure of porcine NORFA using two software (RNAfold and RNAstructuer including SHAPE-map functions). As shown in Fig.R3b, 29 helices and 4 junctions were identified within porcine NORFA but none of which is high conservative among other species (H21 for example, Fig.R3c).
Second, the tertiary structure and the domains of NORFA were predicted using RNAcomposer. Three domains were predicted (D1: 44nt-192nt, D2: 224nt-515nt, D3: 546nt-661nt) and we noticed that three domains just had low conservation among different mammal species (Fig.R3d). In addition, we also analyzed the similarity of the potential open reading frame (ORF, only one ORF consisted by 168 nt was identified in the reversed strand) of porcine NORFA but its conservative was low (<25%) among other species (Fig.R3e). All the data above demonstrate that porcine NORFA only has low structure conservation.
On the other hand, It is worth noting the primary structure (sequence) of genes determine their secondary, tertiary and even space structure. Furthermore, the similarity of gene structure determined the conservation of their domains and functions among different species. To our knowledge, the conservation of lncRNA is usually determined by their primary structure. Besides, we also noticed that in order to analyze the conservation of lncRNAs, it is necessary to analyze their sequence and chromosome locations [1][2][3][4][5] . Thus, the primary conservation and chromosome location of NORFA were analyzed and we found that the primary structure of NORFA has low similarity and the desert region between EDF1 and TRAF2 (two neighbour genes around porcine NORFA) among different species are not conserved (Fig.R3f, g).
According to the findings, we draw the conclusion that NORFA is pig-specific lncRNA. expression of miR-126 and TGFBR2 at RNA level, and also the TGFBR2 and p-SMAD3 protein levels . 4, Authors state this lncRNA has a important role for sow fertility, however, the evidence provided here is not sufficient to draw any conclusion on it.
Response: Thanks. In this study, we have proved that NORFA suppressed porcine GC apoptosis through miR-126-TGFBR2-SMAD3 pathway signaling axis by using gain-or-loss functions. To our knowledge, GC apoptosis is the main cause of follicular atresia in mammal female ovaries [1][2][3] . Furthermore, we have also demonstrated that NORFA is differentially expressed in healthy (with high level of NORFA) and atretic (with relative low level of NORFA) follicles, suggesting that NORFA is involved in sow follicular atresia by inhibiting GC apoptosis. It has been reported that high-prolific pig breeds represent low atretic follicles, while low-prolific breeds show relative high atretic follicles 4 . Together, these results functionally prove that NORFA is an anti-apoptotic factor in porcine GCs which further relate to sow follicular atresia and fertility.
In addition, we also found that the expression level of NORFA in Erhualian (a Chinese famous pig breed with the highest born number record) follicles is higher than that in Large White (a European pig breed with relative low prolific performance) follicles at all stages during follicular development. Furthermore, we have also proved that the 19-bp duplication mutation in the promoter of NORFA leads to its high expression level by recruiting more NFIX, which functions as a transcription factor. Overall, these findings genetically demonstrate that NORFA is a candidate factor closely association with sow fertility.