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

['Bhavesh S. Parmar', 'Animal Physiology', 'Neurobiology', 'University Of Leuven', 'Ku Leuven', 'Leuven', 'Amanda Kieswetter', 'Ellen Geens', 'Elke Vandewyer', 'Christina Ludwig']

Date: 2023-09

Abstract 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.

Citation: Parmar BS, Kieswetter A, Geens E, Vandewyer E, Ludwig C, Temmerman L (2023) azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion. PLoS Biol 21(9): e3002300. https://doi.org/10.1371/journal.pbio.3002300 Academic Editor: René F. Ketting, Institute of Molecular Biology, GERMANY Received: September 12, 2022; Accepted: August 16, 2023; Published: September 15, 2023 Copyright: © 2023 Parmar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Mass spectrometry data are available via Panorama Public via https://panoramaweb.org/azyx1.url. All other relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Fonds Wetenschappelijk Onderzoek Flanders (FWO Flanders, grant G052217N to LT; www.fwo.be), the Katholieke Universiteit Leuven (KU Leuven, grant C16/19/003 to LT; www.kuleuven.be), and by EPIC-XS support to BSP, CL, LT through grant 823839 of the Horizon 2020 programme of the European Union (epic-xs.eu). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: ECM, extra-cellular matrix; FA, formic acid; HDR, homology-directed repair; IRES, internal ribosome entry site; NGM, nematode growth medium; NMD, nonsense-mediated decay; ORF, open reading frame

Introduction Zyxins belong to a subfamily of conserved LIM domain-containing proteins found across eukaryotes and characterized for their role in cell-ECM (extra-cellular matrix) adhesion and cytoskeleton organization [1–4]. Characterized by a proline-rich N-terminus and 3 consecutive LIM domains in their C-terminal region [1,3,5], zyxins regulate actin assembly and remodeling, as well as cell motility [6–9]. In line with this, human zyxin is implicated in stretch-induced gene expression changes via active nuclear translocation [10]. Moreover, it also promotes apoptosis in response to DNA damage [11]. While multiple distinct zyxin proteins are present in vertebrates (for example, Lpp, Trip6, and Zyx), Caenorhabditis elegans contains a unique zyxin gene, zyx-1, with 5 annotated protein isoforms, of which isoforms a and b are predominantly expressed [5,12,13]. There are anatomical differences in isoform expression, with ZYX-1b observed in body wall muscle, pharynx, vulva, spermathecae, and multiple neurons, whereas ZYX-1a mainly localizes to tail phasmid neurons and uterine muscle, and weakly so, body wall muscle [13]. In C. elegans, zyx-1 has been postulated to have a minor role in reproduction, however, the mechanism(s) and isoform(s) involved remain elusive [14,15]. Beyond this, C. elegans zyx-1 is hypothesized to be functionally analogous to vertebrate zyxin, with LIM domains acting as mechanical stabilizers at focal adhesions, and the proline-rich N-terminus involved in sensing muscle cell damage [12]. Previous studies revealed that only the ZYX-1b isoform regulates synapse maintenance and development, while in the context of a dystrophic mutant background, the longer ZYX-1a isoform partially rescues muscle degeneration in an ATN-1-dependent manner, highlighting how not only expression patterns, but also molecular functions are isoform-dependent for C. elegans zyxin [12,13]. At a gene-regulatory level, this use of alternative splicing products and functional diversification of proteoforms for C. elegans zyxin is in line with observations made for several LIM domain proteins across eukaryotes [16]. In general, alternative and overlapping open reading frames (ORFs) arising out of polycistronic mRNA can contribute to posttranscriptional regulation [17–19]. A recent community-wide effort for annotation of such genomic loci categorized overlapping genes based on their initiation and termination codon with respect to the main coding sequence of a given transcript [20]. Of these, ORFs with an upstream start site (upstream or upstream overlapping; uORF and uoORF) often influence the translation of the main coding sequence, based on the evidence of the ones that have been detected and investigated in detail [21–25]. From a more human-centered 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 [22,26], 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. We previously provided mass spectrometric evidence for 467 splice variants and 85 noncanonical gene products, including from polycistronic and ncRNA translation, in C. elegans [27]. Of these, 1 newly discovered gene, azyx-1 (alternative N-terminal ORF of zyx-1), was identified as an 166 amino acid-long protein, translated from the 5′ UTR of zyx-1. In this study, we provide evidence for 2 protein isoforms of azyx-1, one initiating upstream and another downstream of zyx-1 AUG, and both overlapping the proline-rich N-terminus of ZYX-1 long isoforms. To understand whether zyxin proteins could also be regulated by upstream/overlapping ORFs, we here explore functional relevance of azyx-1 and its relation to zyxin in the model organism C. elegans.

Materials and methods Reagents and tools table in Table 1 PPT PowerPoint slide

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TIFF original image Download: Table 1. Reagents and tools table. https://doi.org/10.1371/journal.pbio.3002300.t001 Worm culture All strains used in this study (S1 Table) were cultured at 20°C on nematode growth medium (NGM) plates seeded with E. coli OP50 [58,59]. Molecular biology For azyx-1 localization, 3,474 bp upstream of the azyx-1 stop codon were amplified by PCR from wild-type genomic DNA, along with 558 bp of azyx-1 3′ UTR and fused to 5′ and 3′ ends of mNeonGreen (minus its start-AUG) using HiFi DNA assembly (NEBuilder). The resultant linear transgene was purified (Wizard Genomic DNA purification kit, Promega) and confirmed by sequencing (oligos p001-p006, S3 Table), and injected into wild-type N2 to generate C. elegans strain LSC1959 (see “Transgenesis” and S1 Table). For overexpression and rescue strains (LSC1950, LSC1951, LSC1960, LSC1997, LSC1999, LSC2001, LSC2052, LSC2053, LSC2055; S1 Table), a 757 bp promoter region upstream of azyx-1 was amplified, as were 535 bp of its 3′ UTR. Next, these were fused to 5′ and 3′ ends of a 587 bp synthesized azyx-1 gBlock (Integrated DNA Technologies (IDT); containing all azyx-1 exons and its first intron, with the ATG at the zyx-1a start mutated to CTG) using HiFi DNA assembly (NEBuilder) and confirmed by sequencing (oligos p007-p010, S3 Table). To build the neuronal marker transgene, mCherry was fused to 1,800 bp of the unc-47 promoter region and 497 bp of the unc-47 3′ UTR using HiFi DNA assembly (NEBuilder) and confirmed by sequencing (oligos p011-p016, S3 Table). CRISPR/Cas9-mediated knockout of azyx-1a For the generation of the lst1687 allele, which contains a 27 bp deletion at the beginning of the azyx-1 ORF (positions -182 to -155 upstream of the zyx-1a start site), the dpy-10 co-CRISPR strategy was used with homology-directed repair (HDR) according to Paix and colleagues [60]. The injection mix comprised 2.5 μl of recombinant codon-optimized Cas9 enzyme, 2.5 μl tracrRNA (0.17 mol/l, IDT), 1 μl dpy-10 crRNA (0.6 nmol/μl, IDT), 1 μl of azyx-1 crRNA (0.6 nmol/μl, IDT, S3 Table), 1 μl dpy-10 repair template (0.5 mg/ml, Merck), and 1 μl repair template for azyx-1 containing a 27 bp deletion that encompasses azyx-1a start codon (1 mg/ml, IDT, S3 Table). The mix was micro-injected in the gonads of young adult N2 Bristol wild types (Zeiss Axio Observer A1 with Eppendorf Femtojet and Eppendorf Injectman NI2) [61]. Offspring were screened for the desired CRISPR/Cas9-mediated deletion by SspI (FastDigest Thermo Fisher) cleavage of PCR products of the azyx-1 locus, which is only possible after HDR (oligos p007 and p017, S3 Table). The syb7030 allele was acquired commercially (SunyBiotech) and corresponds to a precise nucleotide exchange of the azyx-1a start codon, wherein the -184th to -182nd basepairs upstream of zyx-1a were mutated from ATG to TAC. The presence of homozygous lst1687 and syb7030 alleles in the azyx-1a mutant strains LSC1898 and PHX7030 (S1 Table) was confirmed via sequencing. Sample collection and preparation for proteomics Adult worms were synchronized by standard hypochlorite treatment [62]. After overnight incubation in S-basal (5.85 g NaCl, 1 g K 2 HPO 4 , 6 g KH 2 PO 4 in 1 L milliQ) on a rotor at 20°C, the L1 arrested animals were grown on NGM plates seeded with E. coli OP50. For wild-type sampling at different ages, we collected worms at larval (L4, 48h post L1 refeeding), day 1 adult (20 h post L4 harvest) and post-reproductive, day 8 of adulthood stages. For day 8 samples, offspring were avoided by supplementing the worm cultures with 50 μl of a 50 μm fluorodeoxyuridine (FUDR) solution every 48 h, as of the L4 larval stage (i.e., L4, and days 2, 4, and 6 of adulthood) [63]. For comparisons of wild types with azyx-1 deletion mutants, both strains were synchronized and then grown until larval stage L4 or day 1 adult stage. For sampling, worms were washed off NGM plates with S-basal and allowed to settle in conical tubes for 10 min. Following that, the supernatant was removed and worm pellets were diluted to 15 ml in S-basal for sorting. Worms were sorted using a Complex Object Parametric Analyzer and Sorter (COPAS) platform (Union Biometrica, Holliston, Massachusetts, United States of America) for each sample individually. Four independently grown populations of worms were used per condition. We collected 200 animals per sample for day 8 or 1,000 animals per sample for all other conditions into a 1.5 ml Eppendorf LoBind tube. The worms were pelleted by spinning at 1,500 g for 1 min, S-basal was removed, and 200 μl of 50 mM HEPES were added to the worm pellet, spun at 1,500 g for 1 min and the supernatant was carefully discarded ensuring no worms were lost in pipetting. Finally, the pellet was supplemented with 1 fmol/worm of synthetic spike-in peptide (EAVSEILETSRVSGWRLFKKIS), comprising a proteotypic peptide for quantitation (EAVSEILETSR) (Vandemoortele and colleagues [64]) fused to a HiBit Tag (VSGWRLFKKIS) via a tryptic cleavage site, from a stock solution in water at a concentration of 100 fmol/μl. The pellet was snap frozen in liquid nitrogen and stored at -80°C until further processing. The duration from initial worm collection off NGM until snap freezing was approximately 20 min and carried out at 20°C. For protein extraction, worm pellets were thawed on ice with 100 μl of lysis buffer (8 M Urea, 2 M Thiourea in 10 mM HEPES) and lysed by sonication using a probe sonicator (40% amplitude, 5 s ON, 10 s OFF × 10). The lysate was spun at 15,000 g for 10 min and the supernatant was transferred to a fresh 1.5 ml Eppendorf LoBind tube. Protein concentration was estimated using a Bradford assay and sample aliquots corresponding to 50 μg of total protein were processed further for LC-MS/MS. For this, each sample was reduced with 5 mM dithiothreitol at 56°C for 30 min and alkylated with 25 mM of iodoacetamide for 20 min at room temperature. The lysate was diluted to 1M urea and digested overnight at 37°C with 2 μg of sequencing-grade trypsin (Promega), after which the sample was acidified to 0.1% formic acid, cleaned using Pierce C18 spin columns as per the manufacturer’s protocol and dried in a Savant SpeedVac. The dried peptides were dissolved to 0.1 μg/μl in 2% acetonitrile/98% H 2 O/0.1% formic acid (FA)/0.1X Biognosys iRT peptides (for retention time calibration). Peptide ion selection for targeted quantification Peptide ions useful for quantification of proteins of interest (ZYX-1 and AZYX-1), and of proteins used for data normalization (GPD-3, HIS-24, spike-in) were selected based on an unscheduled parallel reaction monitoring (PRM) experiment. To accurately normalize data across age and conditions, we chose 3 normalization options: 2 relying on endogenous C. elegans proteins—viz. GPD-3 (GAPDH homolog—4 peptides), HIS-24 (Histone homolog—4 peptides)—and one relying on the externally added synthetic spike-in peptide (1 peptide). Skyline-daily was used to build an initial library [54]. For all proteins of interest, all theoretically predicted tryptic peptides with a length between 7 and 26 amino acids were added to the initial spectral library. In total, 98 peptide precursor ions were selected and measured in an unscheduled PRM experiment that was run on a pooled sample consisting of all peptide samples used in this study and analyzed with Skyline. Subsequently, 23 measured peptide precursors representing 22 peptides and 5 target proteins (ZYX-1, AZYX-1, GPD-3, HIS-24, Spike-in) were selected for the final PRM measurements. Additionally, 11 MS1 ions of the Biognosys iRT reference peptides were included in the precursor list. Details of all peptides and corresponding protein(s), including their uniqueness in the proteome database, can be found in S2 Table. Targeted LC-MS/MS measurements Targeted measurements using scheduled PRM were performed with a 50-min linear gradient on a Dionex UltiMate 3000 RSLCnano system coupled to a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The spectrometer was operated in PRM and positive ionization mode. MS1 spectra (360 to 1,300 m/z) were recorded at a resolution of 60,000 using an AGC target value of 3 × 106 and a MaxIT of 100 ms. Targeted MS2 spectra were acquired at 60,000 resolution with a fixed first mass of 100 m/z, after HCD with a normalized collision energy of 26%, and using an AGC target value of 1 × 106, a MaxIT of 118 ms and an isolation window of 0.9 m/z. In a single PRM measurement, 23 + 11 MS1 peptide ions (see above) were targeted with a 5-min scheduled retention time window. The cycle time was approximately 2.1 s, which leads to about 10 data points per chromatographic peak. Targeted mass spectrometric data analysis PRM data were analyzed using Skyline (version 64-bit 21.1.0.278 and 22.2.0.527) [54]. Peak integration, transition interferences, and integration boundaries were reviewed manually, considering 4 to 6 transitions per peptide. To discriminate true from false positive peptide detection, filtering according to correlation of PRM fragment ion intensities was carried out. For this purpose, an experimental spectral library was built from the PRM data itself, by searching these with MaxQuant and then loading the generated search results back into Skyline. For confident peptide identification, a “Library Dot Product” ≥0.85, as well as a mass accuracy ≤10 ppm (“Average Mass Error PPM”) were required. We also manually verified the correlation between PRM fragment ion intensties and spectra predicted with the artifical intellegence algorithm Prosit [55]. For peptide and protein quantification, chromatographic peak areas were exported from Skyline in MSStats format, and further processing, quantification, statistical analysis, and visualization were performed in RStudio with the MSStats package [56]. For HIS-24, 3 most consistent peptides out of 4 were considered for downstream analysis and peptide FISQNYK was omitted. The data were log 2 transformed, processed as per default MSStats parameters, and visualized using the ggplot2 package of R. For age analysis, data were normalized to spike-in peptide (1 fmol/worm) and L4 samples were used as the reference. For azyx-1a mutant and wild-type comparison, all 3 normalizations were considered (i.e., GPD-3, HIS-24, and spike-in). To evaluate the internal control stability the pair-wise ratios of each combination were calculated based on Vandesompele and colleagues [65] and the equality of variance was evaluated using Levene’s test. The mass spectrometric raw files acquired in PRM mode and the Skyline analysis files have been deposited to Panorama Public (Sharma and colleagues [66]) and can be accessed via https://panoramaweb.org/azyx1.url. Transgenesis For in vivo localization of azyx-1, the lstEx1065 extra-chromosomal array was generated by mixing 25 ng/μl purified linear transgene [azyx-1p::azyx-1+mNeonGreen::azyx-1 3′ UTR] (see “Molecular biology”) with 25 ng/μl coelomocyte-restricted co-injection marker [unc-122p::DsRed] and 50 ng/μl 1-kb ladder (Thermo Scientific) as carrier DNA. This was injected into Bristol wild type (N2) to generate LSC1959 (S1 Table). All genetic overexpressions and rescues of azyx-1 strains were created via injection of 25 ng/μl of linear transgene [azyx-1p::azyx-1(gBlock)::azyx-1 3′ UTR] with 12.5 ng/μl pharyngeal co-injection marker [myo-2p::mCherry] and 50 ng/μl of 1-kb ladder (Thermo Scientific) as carrier DNA unless otherwise noted (S1 Table). For strains transgenically expressing the unc-47p::mCherry neuronal marker (LSC1998 and LSC1999; S1 Table), 10 ng/μl of this linear construct was injected along with 5 ng/μl pharyngeal co-injection marker [myo-2p::mCherry] and 50 ng/μl of 1-kb ladder (Thermo Scientific), with the addition of 10 ng/μl overexpression transgene [azyx-1p::azyx-1(gBlock):: azyx-1 3′ UTR] for LSC1999. Transgenic strains were always confirmed by observation of co-injection marker presence via fluoresence microscopy, followed by PCR and sequencing of the added target sequences. For overexpression of azyx-1 in the zyx-1 reporter background (LSC1870, which expresses mCherry::zyx-1;zyx-1::GFP, S1 Table), the extrachromosomal array (NM3425) was integrated with UV irradiation as per [67] and outcrossed twice with wild type (N2 Bristol). All injections using a Zeiss Axio Observer A1 with Eppendorf Femtojet and Eppendorf Injectman NI2 were performed targeting syncytial gonads of young adults. Confocal imaging Confocal microscopy was performed using either an Olympus FluoView 1000 (IX81) or a Zeiss LSM900 confocal microscope. To obtain synchronized L4 larvae, a timed egg-laying was performed 48 h before imaging, whereas day 1 adults were synchronized by picking L4 larvae approximately 16 h before imaging. Worms were anaesthetized using of 500 mM sodium azide and mounted on 2% agarose pads. Using Fiji [68], resulting z-stacks were analyzed by performing a sum of slices projection and selecting the region of interest (worm or neuron) with the polygon selection tool. In this region of interest, the mean pixel intensity was measured. Manual scoring of muscular filaments Day 1 adults were imaged with confocal microscopy to investigate muscle fiber integrity in control (RW1595, n = 75), overexpressor (LSC2000, n = 52; LSC2055 n = 40) and mutant strains (LSC2001, n = 57). The resulting image files were randomized in Blinder freeware (Cothren and colleagues), visually assessed and scored between 2 qualitative classes of muscle filaments: (1) normal well-organized or (2) mildly damaged or disorganized (Fig 5A), based on previously reported manual scoring parameters [69]. Briefly: muscle fibers displaying tightly organized filaments aligned in a parallel manner were classified as normal. The increased presence of breakage, thinning, or disorganization of individual muscle filaments were classified as disorganized. Fisher’s exact test was used to determine association between strains and muscle integrity. Bonferroni corrections were used for multiple comparisons and adjusted p-values were reported. Burrowing assay Burrowing assays were performed with synchronized day 1 adults and executed essentially as described by Lesanpezeshki and colleagues [32], with minor adjustments. Briefly, 20 gravid adults for each replicate were allowed to lay eggs on a seeded NGM plate for 3 h and subsequently removed while allowing the eggs to hatch and grow at 20°C. After 70 h, worms were washed off the NGM plates with S-basal and transferred onto unseeded NGM plates to induce a starvation response. After 1 h, 30 adult worms were dropped in 50 μl 26% w/v Pluronic F-127 in a well of a Corning Costar 12-well plate and covered with 2.5 ml of 26% w/v Pluronic F-127 (Sigma-Aldrich) at 14°C. After 15 min at 20°C, the Pluronic F-127 had gelated and a droplet of 20 μl 100 mg/ml E. coli HB101 was added on top as a chemoattractant (time = 0). The bacterial droplet was monitored every 30 min for 3 h to calculate a chemotaxis index as the percentage of worms that had cumulatively reached the bacteria (out of the 100% corresponding to n ≥ 30). At each time point of observation, worms that had reached the bacterial pellet were removed to avoid crowding and reburrowing. Statistical significance was determined by two-way ANOVA.

Acknowledgments We would like to thank Prof. Kathrin Gieseler (Université Claude Bernard Lyon 1, France), Prof. Michael Nonet (Washington University in St. Louis, USA) and Prof. Bart Braeckman (UGent, Belgium) for providing strains, and Dr. Marlies Peeters and Dr. Gerben Menschaert (UGent, Belgium) for valuable discussions.

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