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 C. elegans. We observed conservation of alternative open reading frames 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 highlights its impact on C. elegans muscular integrity and locomotion.

Sample collection and preparation for proteomics Adult worms were synchronized by standard hypochlorite treatment (Porta-de-la-Riva et al. 2012). After overnight incubation in S-basal (5.85 g NaCl, 1 g K2 HPO4, 6 g KH2PO4 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 (20h 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 48h, as of the L4 larval stage (i.e. L4, and days 2, 4 and 6 of adulthood) (Mitchell et al. 1979). 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, MA, USA) for each sample individually. Four independently grown populations of worms were used per condition. We collected 200 animals per sample for day-8, or 1000 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 et al. 2016) 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 mins 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 sec ON, 10 sec OFF x 10). The lysate was spun at 15,000g 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% H2O/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 normalisation (GPD-3, HIS-24, spike-in) were selected based on an unscheduled parallel reaction monitoring (PRM) experiment. To accurately normalise data across age and conditions, we chose 3 normalisation options: two 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 (Pino et al. 2020). 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 analysed 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 Supplemental Table. S2. 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 ionisation mode. MS1 spectra (360–1300 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 normalised 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 ~2.1 s, which leads to about 10 data points per chromatographic peak. Targeted mass spectrometric data analysis PRM data were analysed using Skyline (version 64-bit 21.1.0.278 and 22.2.0.527) (Pino et al. 2020). Peak integration, transition interferences and integration boundaries were reviewed manually, considering 4-6 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 (Gessulat et al. 2019). For peptide and protein quantification, chromatographic peak areas were exported from Skyline in MSStats format, and further processing, quantification, statistical analysis and visualisation were performed in RStudio with the MSStats package (Choi et al. 2014). For HIS-24, 3 most consistent peptides out of 4 were considered for downstream analysis and peptide FISQNYK was omitted. The data were log2 transformed, processed as per default MSStats parameters and visualized using the ggplot2 package of R. For age analysis, data were normalised to spike-in peptide (1 fmol/worm) and L4 samples were used as the reference. For azyx-1a mutant and wild type comparison, all three normalisations 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 et al. (2002) 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 et al. 2018) and can be accessed via https://panoramaweb.org/Peu1.url.