Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish

SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.

protein stability) explaining the lack of any phenotype in these mutants (ie, increased NMD or phosphorylation of Upf1)?
7. The brain size of smg9-/-zebrafish was restored by exogenous Smg9 supplementation via smg9 mRNA injection; Does exogenous Smg9 supplementation via smg9 mRNA injection into smg9-/embryos also restore cardiac contraction and premature ageing?
Minor concerns 1.It would be helpful to clarify the working model of Smox-induced premature aging in smg9 mutant.Smox catalyzes the covertion of spermine to spermidine, and spermidine 'has been reported to prevent cell senescence, suppress the occurrence or severity of age-related diseases, and prolong lifespan' (Page 13).At the same time, this reaction produces ROS and acrolein, which promote premature aging.The fact that the authors observed elevated ROS and acrolein levels in smg9 mutant (Fig. 6) but no protective effect of spermine/spermidine treatment (Supplementary Figure 8) suggest that byproducts play a dominant role.Is this the right interpretation? 2. Is there a more quantitative way to measure ß-galactosidase activity than imaging (Fig. 3d to Fig. 3k)?
3. The authors should rearrange the panels in Fig. 1 and Fig. 7 so that the panels appear in the right order (a, b, c, d…) in the main text.4. In Figure 3a, there is an extra space in '(n=2 0)'. 5.In Figure 6a and 6c, both wildtype and smg9 mutant fish were labeled as smg9-/-.
Reviewer #3 (Remarks to the Author): The authors have created a novel smg9 zebrafish mutant that is valuable to study as human mutations in Smg9 result in rare disorders affecting the heart and brain.That being said, the zebrafish mutant does not recapitulate the human disease-causing allele(s), and the human allele(s) are only somewhat described and could be better explained.They present a lot of phenotypic data in this manuscript in addition to some molecular analysis that could underly zebrafish mutant deficits.The overexpression of human Upf1 in zebrafish smg9 mutants is not well controlled for, however, which dampens some enthusiasm for the relevance of this zebrafish model to human disease cases.A lot of the data is well presented in figures, however, I think that throughout some clarification is needed in multiple places and conclusions are incorrectly or overstated for what they are.Lastly, further analysis would strengthen some of these conclusions/results.Detailed comments: -The authors should define SMG9 sooner including in the abstract, title, and provide a bit more about its function/ontology as a kinase inhibitor to strengthen the relevance of the manuscript from the start for readers.
-smg9-/-should be explained sooner as an indel mutation and a proper zebrafish allele name should be created or obtained per zfin.orgguidelines.This will keep things consistent in and available to the field.
-where is smg9 expressed?are there any differences in expression between zebrafish and humans that could explain any phenotypic differences?-The authors do a good job of documenting advanced aging using molecular markers and other indicators, but could it just be that heart and brain malformations underly this and not smg9 specifically?A long term or timed rescue experiment would strengthen the molecular link to aging for smg9 or an alternate explanation should be provided.-The early smox mRNA increase experiment cannot be linked to later aging without making further observations plus use of other molecular tools.Observing acute defects on heart and brain and rescuing these with smox inhibition alone are not enough; observe aging or aging markers later.
-On both Page 4 results "pathological relationship" and Page 8 "pathological examination" the term pathological is not used correctly.
-How much smg9 mRNA was used for the rescue?Methods only state that -"Each mRNA (400 ng/µL) was injected into one-cell-stage embryos" How much per embryo or ng per embryo?-Remove smg8 mutant info, not needed as there was no phenotype observed yet -focus on smg9 only in this manuscript.It does not strengthen the paper, just makes it longer.
-Is the human mutant SMG9 protein remaining or do the authors mean a gene mutation in the human gene on page 6 -it is written as a human protein so this is not clear.-Page 6, needs revision -"Thus, Smg9 deficiency results in impaired cardiac function but not affect morphogenesis in the developing hearts of zebrafish."The sentence is not complete.
-Why is aging and fertility all in supplement figures/data?If aging is a major result as indicated in the title it should be a main figure/main document inclusion and other things removed or moved to the supplement that are less of the main focus.
-Did the authors test to see if hets (smg9+/-) have shorter life or any notable phenotypes since they have a genotyping protocol?-Page 8 -"These data suggest that Smg9 deficiency may lead to premature aging" indicates that the authors lack confidence in these results."May" is used several times throughout this manuscript, and it would be preferable if they could make more definitive conclusions for more of their experiments.
-The B-gal results are disorganized and jump between ages and location (skin v. brain) -need to streamline for understandability of the analysis.-Many genes including smox need to be defined by name and functionally throughout, not just an abbreviation given.
-Supp Figure 7 -what age fish?-No sense made here, Page 10 -"…resulting in negative regulation of the NMD machinery.Thus, SMG9 deficiency is expected to promote the NMD machinery."A negative regulation means suppressed, limited, not promoted.This needs to be fixed.Increased target mRNA in zebrafish then means that NMD machinery IS negatively regulated, not promoted; active NMD would downregulate these transcripts instead -why is this result "contrary" ?This is not clear.
-Could phosphorylation of human UPF1 be different in zebrafish b/c it is fish and not human cells -why not use cell culture to explore this or proper molecular and biochemical controls or co-express human SMG9 with UPF1 in fish?Can human SMG9 rescue smg9-/-to strengthen the human zebrafish connection?-Is smg9 targeted by NMD in zebrafish mutants?Need to confirm LOF and that no partly functioning protein is present especially b/c the human mutation is missense (Page 10 described).This is different than zebrafish created in this study.
-"Notably, ROS production progressively increases with age, consistent with the severity of the disease phenotype."Page 12 -is progressive really just 2 time points observed?See also Fig 6; Progressive indicates more than two samplings.
-How do you separate heart rhythm verses contraction from a florescence image movie?This is not clear it can be done.
-Regarding stats -are all data normally distributed, any outliers?ANOVA may not be best if not a normal distribution.Is a power test done to detect if all sample sizes are big enough?-Fig 3 -regarding positive area of the brain, how was this measured, just across the dorsal surface?Are there better images to show this?Tissue sections would be better at equal positions or across an equal number/size of brain sections.
-Were male verses female differences by 3 mos and beyond done to account for any sex differences in smg9 function and NMD?
Reviewer #4 (Remarks to the Author): Summary: This manuscript sought to define the role of nonsense-mediated decay (NMD) factor SMG9 in zebrafish.The authors used a CRISPR/Cas9 approach to generate a line of mutant fish, which displayed defects in brain size, cardiac contractility, and induced premature aging phenotypes.They attributed these phenotypes to overexpression of the smox RNA, which the authors show is upregulated in SMG9 mutant fish.The results of the study are interesting and novel, however this reviewer finds a conceptual flaw in the design and description of the smg9-/-as a "null", as described below.Please address this and the other concerns outlined below.
Major concern: 1.The authors generated a "frameshift deletion" in exon 1, which they state, "likely corresponded to a null allele".However, the authors fail to experimentally demonstrate this is a null mutation.This is essential to describe the mutants as "null".A search of Ensembl by this reviewer found that Danio rerio has two isoforms for smg9, which differ by the first two exons (absent in coding for one isoform).A frameshift in exon 1, as the authors have done, likely only disrupts one of the two isoforms and may be a hypomorphic allele rather than a null, which may account for the difference in embryonic lethality compared to the mouse knockouts that the authors reference.The authors must address this issue experimentally (for example, using qRT-PCR or Western blot).
Additional concerns: 1. Authors do not adequately address/discuss differences in phenotypes observed between this study and those in mouse (knockout lethal vs viable) and human (function of SMG8, SMG9 as a positive vs negative regulator of UPF1 phosphorylation).

Point-by-point responses to the Reviewers
Thank you very much for reviewing our manuscript.We appreciate the insightful comments of the reviewers.We have provided point-by-point responses to each comment below and made the corresponding revisions to the manuscript document.To make the manuscript revisions easier to locate, they have been highlighted in yellow.

Point-by-point replies to Referee #1
In this paper, the authors focused on Smg9, one of the Nonsense-mediated mRNA decay (NMD) components, and used its knockout zebrafish to clarify the role of Smg9 in the NMD machinery and proposed a new molecular mechanism for the onset of heart and brain malformation syndrome (HBMS) caused by SMG9 mutations.This paper is highly commendable in that it is the first in zebrafish to clarify the role of Smg9 and Smg8 in the NMD mechanism, which could not be analyzed due to the embryonic lethality of Smg9 knockout mice.The authors first showed that Smg9KO fish exhibit abnormalities in brain development, particularly in the terminal brain, and cardiac contractile function, as well as a short-lived, premature aging phenotype.The molecular mechanism of this phenotype was shown to be due to the accumulation of transcripts of NMD target genes.In contrast, Smg8KO did not exhibit this phenotype, and the addition of Smg8KO to Smg9KO did not alter it, indicating that Smg9 plays an essential role in Upf1 phosphorylation and execution of the NMD machinery.This finding overturns the established notion that Smg9 inhibits the NMD pathway.The authors also hinted at transcriptional variation in human

Smg9 mutations. I believe that the paper is concise, to the point, and worthy of publication. The reviewer's main concerns are as follows:
Response: Thank you for your insightful comments and suggestions.We appreciate the opportunity to address the concerns raised by your review.

1) The authors found increased expression of Smox and Gpx3 in Smg9KO, but argue that
the former is due to NMD deficiency and the latter is secondary.
To clarify this point, (i) Is premature aging not observed in Gpx3-transgenic fish?
Response: We recognize the importance of investigating premature aging in Gpx3transgenic fish.However, generating Gpx3-transgenic fish is time-consuming.To expedite our research and provide relevant insights, we checked the brain size at 14 dpf after injecting gpx3 mRNA into single-cell-stage embryos.Injection of gpx3 mRNA significantly decreased brain size (Supplementary Figure 13c, d), suggesting two possibilities: (1) elevated gpx3 expression contributes to the smg9 mutant phenotype, as does elevated smox expression, or (2) elevated gpx3 expression occurs downstream of elevated smox expression and is responsible for the smg9 phenotype.

(ii) Is gpx3 expression enhanced in Smox-transgenic fish?
Response: Investigating whether gpx3 expression is increased in smox transgenic fish is reasonable.However, the generation of smox transgenic fish is time-consuming.To address this concern in a timely manner, we performed mRNA assessments of gpx3 at 7 dpf by injecting smox mRNA into single-cell-stage embryos.No significant upregulation in gpx3 mRNA expression was observed (Supplementary Figure 13a).Thus, rather than being downstream of smox, gpx3 seems to be directly regulated by Smg9 deficiency, similar to smox.The manuscript text has been revised, and a description has been added to the Discussion section (page 20).

(iii) Does Smox inhibitor reduce Gpx3 expression in Smg9KO? should be investigated.
Response: Investigating the effect of a Smox inhibitor on Gpx3 expression in smg9 oi7/oi7 zebrafish is important.We exposed embryos 3 h post-fertilization to 800 μM of the SMOX inhibitor MDL72527 for 3 days and subsequently examined gpx3 mRNA levels.Treatment of smg9 oi7/oi7 zebrafish with Smox inhibitors did not significantly affect gpx3 mRNA levels (Supplementary Figure 13b).Therefore, it is likely that Gpx3 is regulated directly by Smg9 and not downstream of Smox, consistent with the results mentioned in the previous response.
In addition, injection of gpx3 mRNA into wild-type zebrafish decreased brain size, suggesting that the smg9 oi7/oi7 phenotype is not caused solely by increased smox expression but also by increased Gpx3 expression.

2) The rescue effects of Smox and ROS inhibitors on brain development and cardiac function are very interesting, but it is unfortunate that the analysis is limited to some phenotypes. The quality of this paper would be improved if it could be shown whether each inhibitor also exerts effects on other indicators of premature aging (life span, fertility, motility, and beta-gal activity).
Response: We thank you for your constructive feedback and insightful suggestions.
To address your comments, we performed additional experiments to evaluate the effects of SMOX and ROS inhibitors on several indicators of premature aging.Embryos were treated at 3 h post-fertilization with 400 μM MDL72527 and 25 μM N-acetyl-l-cysteine separately for 14 days.This duration was chosen to minimize the potential toxicity associated with prolonged exposure to inhibitors.
No improvement was observed in premature aging phenotypes (brain SA-β-Gal, survival curve, or fertilization rate) after inhibitor treatment (Supplementary Figure 16).This result may have occurred because treatment with inhibitors only in the juvenile stage does not completely suppress the accumulation of smox and ROS.Generating smg9 mutant zebrafish with a smox deficiency would be necessary to test this hypothesis (page 16).However, smox mRNA injection significantly increased SA-β-Gal levels in 4-monthold brains (Fig. 6f), suggesting that the increase in smox mRNA due to Smg9 dysfunction contributes to a premature aging phenotype (page 14).

3) Are there any additive or synergistic effects between Smox and ROS inhibitors, which may be important in considering whether ROS production in Smg9-KO is solely due to increased Smox expression.
Response: We thank the referee for the insightful question regarding the potential additive or synergistic effects of the SMOX and ROS inhibitors.To address this, we treated embryos with 400 μM MDL72527 and 25 μM N-acetyl-l-cysteine for 3 days from 3 h post-fertilization and assessed the brain size at 14 dpf (Supplementary Figure 17).The combination of SMOX and ROS inhibitors was effective in suppressing brain phenotypes.
However, another factor upregulated by Smg9 deficiency may contribute to ROS accumulation.To resolve this issue, we need to identify the factors related to ROS regulation through comprehensive analysis in the future.We have added this information on page 16.We believe these experimental data improve our understanding of the interplay between Smox and ROS inhibitors in ameliorating the phenotypes observed in the Smg9 mutant zebrafish.

Point-by-point replies to Referee #2
In the manuscript, "SMG9 links nonsense-mediated mRNA decay to premature aging in zebrafish", the authors report a novel finding that smg9 knockout in zebrafish triggers premature aging and cardiac defects, similar to pathogenic SMG9 mutations in human patients.The manuscript provides convincing evidence of premature aging both on the cellular and organismal levels in smg9 mutants.Mechanistically, they hypothesize that smg9 knockout impairs nonsense-mediated mRNA decay (NMD), leading to upregulation of spermidine oxidase (smox) and other genes.The byproducts of Smox, i.e., reactive oxygen species (ROS) and acrolein, accumulate in the cell and therefore cause oxidative stress, DNA damage, senescence, and, eventually, premature aging.However, a few major and minor concerns need to be addressed to support these conclusions.
Major concerns 1.The authors introduced a frameshift mutation in smg9 and smg8, respectively, and they predicted these alleles to be null alleles.RT-qPCR and western blot (depending on antibody availability) are necessary to support this hypothesis, or the conclusions need to be tempered.

Response:
We thank you for your thoughtful evaluation of our manuscript and suggestion to provide additional support for the characterization of the frameshift mutations in smg9 and smg8 as null alleles.
The anti-human SMG9 polyclonal antibody from Bethyl Laboratories reacted with the zebrafish Smg9.Therefore, we performed western blot analysis and confirmed the absence of Smg9 protein expression (Fig. 1b).A minor isoform reported in Ensembl was not observed, indicating that there was no expression of this protein isoform in zebrafish (Supplementary Figure 2a).We registered this smg9 mutant zebrafish with ZFIN as smg9 oi7/oi7 and refer to it as such in the revised manuscript (pages 4-5).
We were unable to obtain an antibody that reacted with zebrafish Smg8.Therefore, RT-qPCR was performed to confirm the deletion of the wild-type smg8 mRNA (Supplementary Figure 3c).Because the possibility of hypomorphism cannot be ruled out for smg8 −/− , referring to it as "mutant" rather than "null" is appropriate.It was registered in ZFIN as smg8 oi8/oi8 and is referred to as such in the revised manuscript (page 6).

In humans and mice, SMG9 is known to suppress NMD by inhibiting SMG1 kinase
activity, and, consequently, UPF1 phosphorylation.In this study, the authors showed that smg9 knockout in zebrafish leads to upregulation of NMD prone transcripts (Fig. 4a to Fig. 4j) and decreased Upf1 phosphorylation (Fig. 4k and Fig. 4l).Both findings suggest a positive role for smg9 in NMD.This discrepancy is intriguing.
(i) The authors should consider discussing the conservation of smg9 among different species.
Response: SURF components are highly conserved in vertebrates (Causier et al., Scientific Rep 2017), with more than 70% of the amino acids conserved between zebrafish and humans.Structural analysis of the human SMG1 protein revealed that the domain of SMG important for regulating activity is conserved.We have added this information on page 12-13.
(ii) More importantly, they should consider looking into Smg1 phosphorylation and/or Smg1 activity to strengthen the link between Smg9, Smg1, Upf1, and NMD.
Response: SMG1 activity correlates with UPF1 phosphorylation, with increased UPF1 phosphorylation resulting in increased SMG1 activity.Therefore, SMG1 activity was tested by measuring the degree of UPF1 phosphorylation.
To address the referee's suggestion, we examined whether human and zebrafish SMG8 and SMG9 are interchangeable in the SMG1:SMG8:SMG9 complex.First, we overexpressed the human SMG8:SMG9 heterodimers in 293T cells.As previously reported (Yamashita et al., Genes Dev 2009), the human SMG8:SMG9 heterodimer suppresses SMG1 activity in vitro.This effect was also observed upon overexpression of the zebrafish Smg8:Smg9 heterodimer.Furthermore, human SMG8 and zebrafish SMG9 heterodimers, and conversely, zebrafish SMG8 and human SMG9 heterodimers, inhibited the activity of human SMG1 in UPF1 phosphorylation in vitro (Supplementary Figure 10b).These results indicate that the components of the SMG1:SMG8:SMG9 complex are mutually interchangeable between humans and zebrafish.However, this result conflicts with the decrease in UPF1 phosphorylation in smg9 oi7/oi7 zebrafish observed in vivo (pages 12-13).
The different effects of SMG8/9 on SMG1 activity in vitro and in vivo may be caused by differences in spatial and temporal regulation.SMG9 is involved in the stability of SMG1 and the efficiency with which the SMG8/9 and SMG1 protein complex is recruited to EJC (Arias-Palomo et al., Genes Dev 2011).Indeed, previous reports have shown that NMD is regulated positively by SMG8/9 in vivo, even though SMG8/9 suppresses SMG1 phosphorylation in vitro (page 22).
(iii) The authors should also explain their choice of using overexpressed human UPF1 instead of endogenous zebrafish Upf1 to assess UPF1 phosphorylation in Fig. 4k and Fig. 4l.

Response:
We injected human UPF1 instead of zebrafish Upf1 because we had no antibodies that recognized phosphorylated UPF1 in zebrafish.We confirmed that the antihuman phosphorylated UPF1 antibody did not recognize phosphorylated Upf1 in 293T cells transfected with zebrafish Smg1 and Upf1.We used human 293T cells to test whether zebrafish Smg1 can phosphorylate human UPF1 and found that the phosphorylation of UPF1 in 293T cells was enhanced by zebrafish Smg1 expression (Supplementary Figure 10a).An explanation of this result is provided in the text.An explanation has been added on pages 12-13.

The manuscript focuses on smox because a previous study has shown that 'smox…were
upregulated in patients with the SMG9 homozygous missense mutation (Val184Ala)' (10).

However, this study also showed that SMG9(Val184Ala) does not affect NMD ('…normal SMG9 function may be involved in transcriptional regulation without affecting nonsense mRNA-induced NMD'). Thus, it is necessary to explain the relevance of smox to NMD, e.g., what features render smox mRNA an endogenous NMD substrate.
Response: Thank you for your suggestion to clarify the relevance of smox in nonsense-mediated mRNA decay (NMD).This cellular quality-control mechanism identifies and degrades mRNAs containing premature termination codons (PTCs).In NMD, endogenous mRNA substrates are naturally targeted for degradation.Several features in addition to PTCs, such as a uORF, long 3'-UTR, and alternative splicingproduced PTC, can make mRNA an NMD substrate (Peccarelli and Kebaara, Eukaryot Cell 2014).
The mRNAs containing a uORF or long 3'-UTR are thought to be regulated by the same machinery that recognizes PTC-containing mRNA; however, Rahikkala et al.
reported that endogenous NMD targets are modified in peripheral blood samples in patients carrying SMG9 homozygous missense mutation, although PTC-containing mRNAs were not affected.The uORF structure in human SMOX mRNA is conserved in zebrafish smox mRNA, suggesting the smg9 mutation increases smox mRNA by the same mechanism as in patients.
We did not perform a comprehensive expression analysis of the changes in the expression of PTC-containing alleles in Smg9 oi7/oi7 ; however, PTC-containing mRNAs might not be affected in Smg9 oi7/oi7 as seen in human patients.The reason for the absence of changes in PTC-containing mRNA in patients with SMG9 mutations remains unclear.
The relatively mild decrease in NMD activity caused by the hypomorphic missense mutation in human SMG9 may have a greater effect on these endogenous targets.We have added this information to the Discussion (pages 20-21).7, the authors used SMOX or ROS inhibitors to attempt at rescuing the brain and cardiac defects in smg9 mutants.However, some of these effects are mild and not significantly different between smg9 mutants with and without inhibitor treatment (Fig.

4c-d, e-f, g-h and k-l). Have the authors tried generating smox-/-; smg9-/-mutants?
Response: For those that were not statistically significantly different with and without inhibitor treatment, we have revised the text to read "tendency to be restored" (page 16).
We agree that it would be best to create and validate smox -/-and smg9 -/-double mutants, as suggested by the referee.However, considering the time constraints associated with generating double mutants of smox -/-and smg9 oi7/oi7 , we adopted an alternative approach employing smox morpholino knockdown.As shown in Figure 8m-r, significant phenotypic improvement was observed with the inhibitor treatment.In addition, more defined improvements were observed in simultaneous treatment with Smox inhibitors and NAC (Supplementary Figure 17) (page 16).

Figure 6 clearly showed signs of oxidative stress in the brain of smg9 mutant. Is this also the case in the heart, which then causes cardiac defect? To establish a causal relationship between Smox and the observed phenotypes in different organs, tissuespecific approaches should be taken, such as measuring smox expression levels in dissected tissues and genetically manipulate smox in a tissue-specific manner.
Response: We appreciate the reviewer's insightful comment and the importance of investigating the potential causal relationship between increased Smox expression, oxidative stress, and the observed cardiac defects in the smg9 mutant.
In response to the referee's suggestion, we measured the expression levels of Smox in various smg9 -/-zebrafish tissues.Our analysis revealed that elevated smox mRNA levels were observed in the brain, muscle, eye, heart, intestine, and testes of smg9 mutants, suggesting that the oxidative stress associated with increased Smox expression is not confined to the brain (Supplementary Figure 12).The brains and hearts of larvae exhibit particularly noticeable phenotypes indicative of their greater susceptibility to ROS.We have added this discussion (page 18-19).
This observation strongly suggested a potential association between elevated Smox expression and oxidative stress in cardiac tissues, providing a plausible mechanism for the observed cardiac defects.To further explore the tissue-specific effects of Smox and its role in cardiac defects, we need to conduct tissue-specific genetic manipulations of Smox, as suggested by the referee.This approach will provide valuable insights into the direct effect of Smox on cardiac function and help establish a causal relationship between Smox, oxidative stress, and the phenotypes observed in different organs.

As smg8 null mutants do not exhibit any phenotype and smg8 deficiency in smg9 -/-
zebrafish did not affect the phenotype of smg9-/-; Are these results suggesting that either Smg8 or Smg9 can suppress the kinase activity of Smg1 until the Smg1-Upf1 complex joins the EJC, whereas only Smg9 plays a role in promoting the phosphorylation of Upf1?

Could Smg9 compensate for the loss of Smg8 in the smg8-/-mutants (ie, increased transcription or protein stability) explaining the lack of any phenotype in these mutants (ie, increased NMD or phosphorylation of Upf1)?
Response: No difference in the phenotype being observed between smg9 -/-and smg8/9 double-KO is reasonable because a biochemical and structural study revealed that SMG8 binds to SMG1 via SMG9 (Fernandez et al., Nucleic Acids Res 2011).However, the lack of a phenotype in smg8 -/-zebrafish was surprising.
As the referee proposed, Smg9 might compensate for Smg8 deficiency because the NMD pathway is characterized by an autoregulatory negative feedback mechanism that regulates the expression of core NMD factors through the NMD machinery (Huang et al., Mol Cell 2011).Therefore, we examined smg9 mRNA levels in smg8 -/-zebrafish, finding a notable decrease in smg9 mRNA levels compared with that in wild type (Supplementary Figure 5), suggesting that Smg9 does not compensate for a Smg8 deficiency.
The interaction between Smg8 and Smg9 in regulating Smg1 activity and Upf1 phosphorylation is an intriguing aspect that warrants further investigation.We understand that SMG8/9 suppresses the activity of free SMG1 until its recruitment to the EJC, where SMG9 contributes to the stabilization of SMG1 protein, similar to the regulation of p110 of the same family, PI3K, by p85 (Fox et al., Biochem Soc Trans 2020).
Although SMG1 activity is regulated mainly by SMG8, SMG9 might contribute more to SMG1 stabilization than SMG8 (Arias-Palomo et al., Genes Dev 2011).After recruitment to the EJC, it is possible that SMG9, rather than SMG8, positively regulates the activity of SMG1.SMG9 alone does not suppress SMG1 kinase activity in vitro (Deniaud et al., Nucleic Acids Res 2015).Indeed, we observed that overexpression of SMG9 alone in HEK293T cells did not suppress UPF1 phosphorylation, unlike the cooverexpression of SMG8/9 (Supplementary Figure 10b and data not shown).
In addition, PTC-containing mRNAs and other endogenous targets of NMD could be regulated by different mechanisms of the NMD pathway.SMG8 may be required for regulating PTC-containing mRNA, but SMG9 may contribute more to regulating the other endogenous targets of NMD.However, this requires clarification in future studies.
These factors may explain the lack of a phenotype in smg8 mutants.We have added this information to the Discussion section (pages 21-22).
7. The brain size of smg9-/-zebrafish was restored by exogenous Smg9 supplementation via smg9 mRNA injection; Does exogenous Smg9 supplementation via smg9 mRNA injection into smg9-/-embryos also restore cardiac contraction and premature ageing?
Response: We appreciate the referee's interest in understanding the broader effects of exogenous Smg9 supplementation on cardiac contraction and premature aging in smg9 oi7/oi7 zebrafish.To address this, we injected zebrafish smg9 mRNA into single-cellstage embryos, evaluating cardiac contractions at six days post-fertilization and premature aging phenotypes in adult fish (Fig. 2g and Supplementary Figure 8).Although exogenous Smg9 supplementation via smg9 mRNA injection into smg9 oi7/oi7 embryos restored the brain and heart phenotypes at the larval stage (Fig. 2g) (page 7), it did not ameliorate premature aging phenotypes such as lifespan, fertility, and β-galactosidase activity (Supplementary Figure 8).Because the premature aging phenotype in smg9 oi7/oi7 zebrafish is observed at the adult stage, long-term expression of exogenous Smg9 is required to elucidate the effect of exogenous Smg9 expression on premature aging phenotypes.The results of these experiments are presented in the revised manuscript (page 10-11).

Minor concerns 1. It would be helpful to clarify the working model of Smox-induced premature aging in smg9 mutant. Smox catalyzes the covertion of spermine to spermidine, and spermidine
'has been reported to prevent cell senescence, suppress the occurrence or severity of agerelated diseases, and prolong lifespan' (Page 13).At the same time, this reaction produces ROS and acrolein, which promote premature aging.The fact that the authors observed elevated ROS and acrolein levels in smg9 mutant (Fig. 6) but no protective effect of spermine/spermidine treatment (Supplementary Figure 8) suggest that byproducts play a dominant role.Is this the right interpretation?
Response: We appreciate the reviewer's insightful observation.To make it more understandable for the reader, we have modified the text as follows (pages 15-16).The smg9 mutant displayed high ROS and acrolein levels (Fig. 7).However, no protective effects of spermine/spermidine treatment were observed (Supplementary Figure 15).These results suggest that byproducts play a dominant role in the premature aging phenotype.

2.
Is there a more quantitative way to measure ß-galactosidase activity than imaging (Fig. 3d to Fig. 3k)?
Response: While our current approach relies on imaging for qualitative assessment, we recognize the importance of incorporating a quantitative method for comprehensive analysis.To improve the quantitation of β-galactosidase activity, frozen sections were used for brain and β-galactosidase staining, providing a more accurate representation of the positive brain areas (Fig. 4g-l and Supplementary Figure 6) 3. The authors should rearrange the panels in Fig. 1 and Fig. 7 so that the panels appear in the right order (a, b, c, d…) in the main text.

Response:
We apologize for the inadequacies in the figure panels.We have corrected them.3a, there is an extra space in '(n=2 0)'.

In Figure
The authors have created a novel smg9 zebrafish mutant that is valuable to study as human mutations in Smg9 result in rare disorders affecting the heart and brain.That being said, the zebrafish mutant does not recapitulate the human disease-causing allele(s), and the human allele(s) are only somewhat described and could be better explained.They present a lot of phenotypic data in this manuscript in addition to some molecular analysis that could underly zebrafish mutant deficits.The overexpression of human Upf1 in zebrafish smg9 mutants is not well controlled for, however, which dampens some enthusiasm for the relevance of this zebrafish model to human disease cases.A lot of the data is well presented in figures, however, I think that throughout some clarification is needed in multiple places and conclusions are incorrectly or overstated for what they are.
Lastly, further analysis would strengthen some of these conclusions/results.

Detailed comments:
1) The authors should define SMG9 sooner including in the abstract, title, and provide a bit more about its function/ontology as a kinase inhibitor to strengthen the relevance of the manuscript from the start for readers.

Response:
We appreciate the referee's thoughtful comments.We have revised the text so that the reader has a better understanding of SMG 9 as follows: Title: Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish Abstract: SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts.
Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS).However, the molecular mechanism underlying HBMS remains unclear.
2) smg9-/-should be explained sooner as an indel mutation and a proper zebrafish allele name should be created or obtained per zfin.orgguidelines.This will keep things consistent in and available to the field.

Response:
We have registered our smg9 and smg8 mutant zebrafish in ZFIN as smg9 oi7/oi7 and smg8 oi8/oi8 , respectively, and refer to them as such in the revised manuscript (pages 4, 6).
3) where is smg9 expressed?are there any differences in expression between zebrafish and humans that could explain any phenotypic differences?
Response: We appreciate the referee's interest in the expression of smg9.To address this, we analyzed smg9 mRNA expression in various zebrafish tissues at 3 months of age and compared it with human tissues (Supplementary Figure 1).We hope this supplement will provide a more comprehensive understanding of smg9 expression in different species.
Except for the testes, the SMG9 mRNA expression patterns were approximately the same, suggesting the phenotypic differences between zebrafish and humans are not due to differences in gene expression.Given that mutations in human patients result in a missense mutation rather than a null mutation, as in our smg9 oi7/oi7 model, a residual hypomorphic function may still be present (page 4).molecular link to aging for smg9 or an alternate explanation should be provided.

Response:
We agree with your suggestion.Although SMOX and ROS inhibitor treatment ameliorated the brain and heart phenotypes in smg9 oi7/oi7 larvae, it did not ameliorate premature aging phenotypes, such as lifespan, fertility, and β-galactosidase activity (Supplementary Figure 16).Timed rescue of Smg9 expression measured by injecting smg9 mRNA into single-cell embryos also did not rescue the premature aging phenotype in smg9 oi7/oi7 zebrafish (Supplementary Figure 8).Response: To address this concern, we performed an additional experiment in which zebrafish smox mRNA was injected into single-cell-stage embryos.We observed and assessed aging markers, including lifespan and fertility, in adult fish to gain a better understanding of the long-term effects of Smox modulation (Fig. 6f, Supplementary Figure 14) (page 14).Because overexpression of Smox during only the larval stage increases SA-β-gal staining in the brains of adult fish, accumulation of ROS during the larval stage should be sufficient to induce an early aging phenotype (Fig. 6f) (page 14).
We hope this additional analysis clarifies the link between early molecular changes and later-aging outcomes in our study.However, the lifespan and fertility of smox mRNAinjected zebrafish were similar to those of the control group, indicating that short-term smox expression is insufficient to induce full aging phenotypes.Investigating whether Smox upregulation is required for the premature aging phenotypes in smg9 oi7/oi7 zebrafish has an experimental limitation because long-term treatment with SMOX and ROS inhibitors is impossible due to the toxicity of these inhibitors.Generating smg9 oi7/oi7 zebrafish with a smox deficiency would be useful for testing this hypothesis.
6) On both Page 4 results "pathological relationship" and Page 8 "pathological examination" the term pathological is not used correctly.
Response: Regarding the term "pathological" on pages 4 and 9, we have changed the text as follows: "To clarify the pathophysiological relationship between HBMS and SMG9, we generated smg9 mutant zebrafish using CRISPR/Cas9 genome editing" (page 4).On page 9, the text was revised to "Histological examination of the testes revealed a decreased number of mature sperm cells in smg9 oi7/oi7 zebrafish." 7) How much smg9 mRNA was used for the rescue?Methods only state that -"Each mRNA (400 ng/µL) was injected into one-cell-stage embryos" How much per embryo or ng per embryo?
Response: We apologize for the inadequate description in the Methods section.We have added the following statement (page 26): Forty picograms of each mRNA were injected into individual embryos at the singlecell stage.
8) Remove smg8 mutant info, not needed as there was no phenotype observed yet -focus on smg9 only in this manuscript.It does not strengthen the paper, just makes it longer.
Response: We appreciate the reviewer's suggestion.However, following the editor's guidance, we have retained the smg8 mutant data.

9) Is the human mutant SMG9 protein remaining or do the authors mean a gene mutation
in the human gene on page 6 -it is written as a human protein so this is not clear.

Response:
We apologize for the errors in the description.We have changed SMG9 to SMG9 (page 7).
10) Page 6, needs revision -"Thus, Smg9 deficiency results in impaired cardiac function but not affect morphogenesis in the developing hearts of zebrafish."The sentence is not complete.

Response:
We apologize for the grammatical error.We have corrected it as follows (page 7): Thus, Smg9 deficiency results in impaired cardiac function but does not affect morphogenesis in the developing hearts of zebrafish.
Smox catalyzes the conversion of spermine to spermidine, and spermidine prevents cell senescence, suppresses the occurrence or severity of age-related diseases, and prolongs lifespan (Al-Habsi et al., Science 2022, Ni & Liu, Aging Dis 2021).At the same time, this reaction produces ROS and acrolein, which promote premature aging.
RT-qPCR of SMG9 mRNA levels in zebrafish and human tissues.

11 )Figure 3 . 12 )
figures have been restructured, and the aging phenotype of the testes is now presented in Figure3.
Thus, heart and brain malformations at the developmental stage do not seem to underlie premature aging phenotypes in adults.Crossbreeding experiments with transgenic zebrafish expressing Smg9, specifically in the brain and heart, will help elucidate the relevance of brain and heart dysfunction in premature aging (pages10-11 and 16,19).
5) The early smox mRNA increase experiment cannot be linked to later aging without making further observations plus use of other molecular tools.Observing acute defects on heart and brain and rescuing these with smox inhibition alone are not enough; observe aging or aging markers later.