azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5′ region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.


General Statements [optional]
The newly identified azyx-1 ORF was named peu-1 in the initial submission of this manuscript, a name that was under consideration with WormBase, who supervise nomenclature of C. elegans genes. In consultation with WormBase, the locus was named azyx-1 instead (the final decision being "azyx-1 will be attributed to F42G4.11. It will be released in WS287 at the beginning of 2023"). We updated this nomenclature in our submission files, including in reviewer comments pasted below. Please note that other than this, no changes whatsoever were made to the reviewer comments.
2. Description of the planned revisions REV #3: Specific thoughts for consideration: Figure 5, Moderate is really minor/moderate with other metrics, and severe is definitely moderate with other metrics. Thus, I'm not sure if normal vs. moderate is needed. This really is a minor point as it doesn't impact results/overall story/importance. This was also pointed out by reviewer #1. We will rename classification more mildly so. REV #1 Fig. 5 Even the 'severe' muscle disruption is quite mild (say, in comparison to loss of talin). Perhaps rephrase these categories? The moderate and severe categories also do not look different to me. Show what the muscle cells look like in zyx-1 deletion and overexpression animals. Is there a way to use quantitative imaging to score these? Can azyx-1 phenotypes be rescued or enhanced by expression (or RNAi) of zyxin in the muscle? Also, clarify what age animals are being tested in the muscle and burrowing assay. We agree and will rename the classes in milder terms. Qualitative scoring (which was done blinded) is the standard in the field as was done according to Dhondt et al. (2021 Dis Model Mech). When tested for muscle integrity and burrowing capacity, animals were day 1 adults. This is mentioned in the Methods section of the current manuscript and will also be included in the captions of the revised figures. REV #2: I am not convinced by the data presented in Figure 5. There does not seem to be much to distinguish the five genotypes, but I concede that I am not used to looking at this type of data. But why was the muscle phenotype not also examined in the azyx-1 rescue lines? Because other reviewers that are familiar with these data point out that the observed differences of panels A-B are indeed milder that what is usually seen, we will rename classifications in the manuscript (see responses above). Because the azyx-1 deletion mutant does not differ from controls in the muscle phenotype, there is no phenotype to rescue for this readout, and no rescue strains were generated. We are not sure what the reviewer may struggle with in (assumedly) panel C (~'to distinguish the five genotypes'). The positive control (zyx-1) behaves as expected in the burrowing assay, with our own mutants within that range, also as expected. All data were scored blinded to avoid any bias and statistical analysis supports the interpretations, all granting confidence to the observed differences. However, because reviewer#3 also would prefer another representation of the data shown in this panel (see below), we will provide an updated panel representation in the revised manuscript. REV #3: Figure 5C-Hard to read. Would displaying lines/tragectories make it easier to understand? Would displaying as violin plots for each timepoint/condition make it easier to visualize? Basically in black and white and in color this is hard to visually process. We will work on another representation for the revised manuscript, since reviewer2 also seemed to struggle with this panel representation.
REV #1: Fig. S2 Match font sizes on Y-axes. Also, indicate any statistical differences and statistics used.  REV #2: I am not convinced by the "overexpression" experiments. These are not well controlled, since no evidence is presented that AZYX-1 is being overexpressed in these lines. Also, since we know that extrachromosomal transgenic lines are highly variable, one would need to test the effect of several independent lines to ensure that the effects that the authors observe are indeed associated with AZYX-1 overexpression and not simply an idiosyncratic effect of the genetic background of a given strain. Finally, there does not seem to be an obvious mechanism by which overexpression of AZYX-1 can impact ZYX-1 function. That doesn't rule out an effect, but based on the data as it is, it is premature to propose such a mechanism. The authors need to show that multiple overexpression lines do reproducibly overexpress AZYX-1 and that this results in reproducible effects of zyx-1 phenotypes. The extrachromosomal strains are indeed variable, but because the background is wild type (in contrast to a deletion mutant background for rescue strains), an overdose of the target provided is expected. As requested in the cross-consultation reviewer communication, we will include quantitative data in our revised manuscript that shows that the used strains (LSC1950, LSC1960, LSC2000) indeed are overexpressors.
REV #2: The data presented in Figure 4F needs to be quantified using the same format as was presented in Figure 4B. Due to the different genetic background of the strains, this is not possible in the exact same way (the red signal of LSC1998 & LSC1999 is not unique to zyxin). We understand that in essence, the reviewer would like us to include a more quantitative representation of these data, and will update the figure accordingly.
REV #2: What is the difference between the overexpression transgenic lines and the "rescuing" transgenic lines? In the Materials and Methods, the same concentration of plasmid was used in injections -so these likely give the same approximate level of transgenic expression. The genetic background: a rescue line adds wt DNA back to a mutant background, while in an OE strain it is added into a wt background. While this can already be derived from the genotype details in Supplemental Table S1, we apologize for not specifying this in the methods section, as it is common practice in the field. These specifications will be added to the revised manuscript.
REV #2: I am not clear what features are being used to characterise the myofibril structures into the three categories. Can the authors annotate the images to indicate the diagnostic features?
The reviewer is correct that manual classification is rather poorly defined in general, which is why it is scored blinded (here as per Cothren et al., 2018 Bio Protoc). We adhered to the reference images by Dhondt et al. (2021, Dis Mod Mech) with visual assessment based on how tightly organized (~parallel) myofilaments are organized, assessing overall increases of bends or breaks in individual myofibers as leading to a less aligned pattern (cf. Fig. 1 of Dhondt et al.). We will add this information more explicitly to the Methods section of the revised manuscript.
3. Description of the revisions that have already been incorporated in the transferred manuscript REV #2: The authors state that there is evidence for synteny and coding region conservation. The data supporting this assertion is not well presented. Presentation and analysis of multiple sequence alignments of the putative homologues involved would strengthen the assertion of synteny considerably. We apologize if the reviewer misunderstood: we discuss likely syntenic conservation, not coding region conservation. The latter is not mentioned in our manuscript, and in fact not convincing indeed. This is not surprising given the bigger sequence diversity observed at the N terminus of zyxins and the partial overlap of these coding sequences, and in line with observations of several others in the RiboSeq community that many identified uORFs are conserved between orthologous genes, but poorly conserved at the amino acid level (e.g. community-driven communication by Mudge et al., BioRxiv 2021 and references therein).
REV #2: The authors are oddly coy about the molecular details of the 27 bp deletion used to study the loss of azyx-1 function. In the absence of these details, it is not possible to assess the validity of these experiments. We need to be given the full molecular details of the allele -precisely which nucleotides are deleted? And how do they affect the coding regions of zyx-1 and azyx-1? I am also confused about why the authors made a deletion allele rather than mutating the AUG of AZYX-1? This would be a cleaner experiment to interpret. Based on the data presented, there are two possible interpretations in addition to the one suggested by the authors: 1) the 27 bp deletion impacts zyx-1 expression due to its impact on the zyx-1 coding region (the coding regions of azyx-1 and zyx-1 overlap); 2) the deletion mutation deletes critical transcriptional control elements. A simpler mutation of the azyx-1 AUG via CRISPR might allow them to rule out the possibility that they have simply compromised a transcriptional control element or damaged the coding region of ZYX-1.
As mentioned above and as will be included more clearly so in the revised manuscript: the deletion is 182-155bp (27bp) upstream of the zyx-1a start site. This was a mutant that could easily be generated via CRISPR, so we proceeded with this one. This edit rules out option1 (there is no change of the zyxin coding region), but (as also considered but addressed differently in the manuscript; see below) retains alternative interpretation 2. There are no regulatory regions or transcription factor binding sites known for the (a)zyx-1 locus (verified in current WormBase version WS285), but that does certainly not fully rule out the possibility either. Rather than creating a series of azyx-1 mutants, be they SNP or small deletion mutants, that would suffer from the exact same duality in possible interpretation, we chose to combine the deletion mutant with rescue and overexpression strains. Because these latter strains do not affect the endogenous zyxin regulatory region, they add far more credibility to the interpretation, than alternative mutants in the azyx-1/zyx-1 locus would.
REV#2. The narrative flow of the introduction could be improved by the judicious use of paragraphs. Line 12, for instance is a clear paragraph break, as is line 24. done

Revision Plan
REV #3: Specific thoughts for consideration: 3) Could more be said about overlapping genes/regulation in humans? Again, not critical _but_ this is such a great piece of work that it would be useful to guide human subjects researchers as to how to best further your work. It is unclear whether the reviewer would like to see an extended introduction and/or discussion. We tried to meet this request without drifting too much from the focus of our current communication by adding the following to the introduction (lines 41-47 of the current draft): "From a more human-centred future perspective, uORFs are a rather unexplored niche for translational research: with a predicted prevalence in over 50% of human genes and first examples regulating translation of disease-associated genes already emerging (Lee et al. 2021;Schulz et al. 2018), the field is bound to not only lead to more fundamental, but also application-oriented insights.
Keeping this broader context in mind, we here focus on more fundamental principles of uORFs in a model organism context.".
4. Description of analyses that authors prefer not to carry out REV#1: Does azyx-1 have zyx-1-independent functions or other regulatory targets? This is an interesting question that is not yet addressed. While this is possible, it is beyond the scope of our current communication. Since the reviewer does not request anything concrete, we would prefer to leave this for follow-up research. While this notion is included in the manuscript, we are happy to more explicitly address this question in the discussion as well.
REV#1: Do the burrowing assay results reflect a neuronal or a muscle function for AZYX-1? Or both? Our manuscript indeed does not yet delve into tissue-specific actions of this newly discovered ORF. While interesting, and in line with reviewer #3's remark, this would be valuable for follow-up research, but is beyond the scope of our current communication. We will make sure the concept is clearly mentioned in the discussion of our findings. REV #3: Specific thoughts for consideration: 1) Could more be done/said about neruo vs, muscular effects of azyx-1 and zyx-1. I appreciate this is beyond the scope of the present manuscript and therefore does not require response if you don't have data or it makes telling the story you want to tell more difficult.
We agree with the reviewer that spatially resolving some of these observations would be a next interesting step, which indeed is beyond the scope of our current communication.

CROSS-CONSULTATION COMMENTS
The following is a conversation among the three referees: REFEREE #2: I appear to be the dissenting voice in terms of concern about the details of the 27 bp deletion and the "overexpression" constructs. I would be interested to know your opinions regarding my comments on these issues. REFEREE #1: I think adding the details of the 27 bp deletion is a reasonable request. It is probably not possible to disambiguate entirely the two effects of the deletion, and changing the start codon may result in an alternate start with other downstream effects. I think just explaining it more fully in the methods would satisfy my concerns. The point about ability to judge conservation is also reasonable but until the theory is firmly out there it is hard to test the conservation and broader applicability to other genes/proteins. Thus, while asking for additional information on these issues is reasonable I do not see the inability to address beyond highlighting as limitation in the text as critical to the overall validity of the work. REFEREE #2: I disagree with Reviewer #3, without knowing the details of 27 bp deletion the most reasonable interpretation of the data is simply that it is a loss-of-function allele of zyx-1. This goes beyond "technical" -at present there is no unequivocal evidence that azyx-1 has any functional significance beyond that it is expressed as a peptide. REFEREE #3: I've been back through the manuscript. They have sequenced the deletion and therefore should be able to provide that information to satisfy the issue(s). For the over expression, short of silencing my experience is that they do over express and when you have multiple lines some express more than others (and some silence more than others). If you want evidence that the peptide is over expressed ask them to quantify via mass spec if it isn't tagged and they can't do a Western. Clearly they have work leading expertise in quantitative mass spec proteomics in C. elegans and should be able to do that. Generally speaking, rescue of a deletion is a pretty good sign that the expression is working though (and is an accepted standard). We apologize if this was not clear from the manuscript, and will clearly include the details in the Methods section: the deletion is 182-155bp (27bp) upstream of the zyx-1a start site, at AT|G+26|TTC. This was confirmed by sequencing; the oligos used for this are listed in table S3 of the manuscript. We address the confusion of rescue and overexpression above, in response to reviewer #2 (who echoes this confusion here).