To explore the role of Naa10 in development, most analyses were carried out using our Naa10 KO model mice that had been generated previously (Yoon et al., 2014) using a targeting vector deleting Exon1, including the start codon, and Exon2 to Exon4 containing the GNAT domain including the acetyl-CoA binding motif, which is crucial for Naa10 function. We also generated another Naa10-deficient mouse, which we called Naa10^tm1a, expressing β-galactosidase rather than the Naa10 gene (Figure 1—figure supplement 1A). Naa10 expression was deficient in Naa10^tm1a mice (Figure 1—figure supplement 1B, C). Especially strong β-gal staining was observed during embryonic stages in the brain, heart, and spinal cord (Figure 1—figure supplement 1D). Male Naa10 KO (Naa10^-/Y) embryos displayed mild to severe developmental defects compared to wildtype (WT) (Naa10^+/Y) embryos. Some Naa10^-/Y mice had lower levels of somites and developmental delay. Additionally, some Naa10^-/Y embryos had a normal number of somites but were retarded in growth (Figure 1A). Some of the embryos underwent lysis or remained arrested at an earlier stage than embryonic day 10.5 (E10.5), with no turning, an abnormal trunk, and underdeveloped facial features. These phenotypes also reproduced in Naa10^tm1a/Y embryos. Next, we assessed whether Naa10 is essential for viability and counted the Mendelian ratios. Both Naa10^-/Y and Naa10^tm1a/Y mice were under-represented after birth, while there was no significant reduction in the embryonic stage in both mouse lines (Supplementary file 1a, b). We monitored the pups daily at postnatal day 0 (P0) to postnatal day 3 (P3) and beyond, and the survival rate of Naa10^-/Y mice dramatically decreased relative to either WT (Naa10^+/Y and Naa10^+/+) or heterozygous female (Naa10^+/-) mice after the first few days of life (Figure 1B), and a few Naa10^-/Y mice with postnatal lethality exhibited severe developmental defects such as craniofacial anomaly, an undeveloped lower body, whole-body edema, and ocular malformations (Figure 1C).

Congenital heart defects are one of the main causes of infant lethality, and cardiac diseases are a common developmental anomaly in OS patients (Casey et al., 2015), with some OS males dying in infancy with cardiac arrhythmias (Rope et al., 2011). Therefore, we investigated whether Naa10 KO affects cardiac development. Development of a four-chambered septated heart is normally complete at E14.5; therefore, we examined the cardiovascular system at E14.5. We identified ventricular septal defects (VSDs) in several Naa10^-/Y embryos, as well as concomitant double outlet right ventricle (DORV) at E14.5 (Figure 1D, upper). VSDs and atrial septal defects (ASDs) were also observed at E18.5 (Figure 1d, bottom), and persistent truncus arteriosus (PTA) or DORV, along with concomitant membranous and muscular VSDs, were found in several of the mice that died in the first day of life (n = 6/28 examined). Given the presence of outflow tract defects and VSDs, we examined whether the ductus arteriosus had closed appropriately or not at birth. Significantly, both Naa10^-/Y and Naa10^-/- females (n = 3/28 examined) exhibited a patent ductus arteriosus, meaning that there is a failure of the mutant in utero cardiovascular system to adapt to adult life (birth) and close the interatrial and aorta-pulmonary trunk shunts that are required for normal fetal life (Conway et al., 2003). As murine outflow tract and VSDs are not compatible with postnatal survival (Conway et al., 2003), these data suggest that congenital heart defects in Naa10^-/Y mice may explain some of their neonatal lethality (Figure 1—figure supplement 2). We also examined surviving adult mice for any possible situs inversus, but we did not observe this in any adult (>4 weeks) Naa10^-/Y mice examined (n = 19). Combined, these data suggest that Naa10 mutant CHDs are mainly confined to aberrant remodeling of the great vessels of the heart, leading to pulmonary overload at birth resulting in lethality.

Some of the surviving homozygous mice (Naa10^-/Y and Naa10^-/-) had reduced body weight (Figure 2A). This reduced body weight continued through weaning, and some mice lost more weight as they developed progressive hydrocephaly. We observed that the smallest weight animal between the Naa10^+/Y and one Naa10^-/Y genotypes was almost always the Naa10^-/Y genotype when the analysis was restricted to only include litters in which there was at least one of each of those genotypes living beyond 4 days of life. For example, 13 litters met this criteria from the mating (Naa10^+/- × Naa10^+/Y), and 12/13 of the litters had the Naa10^-/Y as the lower weight (Fisher's exact test, two-tailed, p-value<0.0001). Five litters met this criteria from the mating (Naa10^+/- × Naa10^-/Y), and of these, all of them had the Naa10^-/Y as the lower weight (Fisher's exact test, two-tailed, p-value=0.0079). Therefore, despite the known variability in weight data as a function of genetic background, environment, and stochastic variation (Pun et al., 2013), it does appear at least for ‘within-litter’ analysis that Naa10^-/Y males are born at a smaller weight than Naa10^+/Y males and on average remained the smallest male in the litter throughout their life.

Although piebaldism has never been reported in humans with OS, all (100%) of the Naa10^-/Y and Naa10^tm1a/Y mice exhibited hypopigmentation on their belly (Figure 2B, upper), with this piebaldism quite varied in its extent but not appearing to correlate in any way with other phenotypes, such as hydrocephaly. Another phenotype with complete penetrance was bilateral supernumerary ribs (14 pairs of ribs instead of 13) in all Naa10^-/Y and Naa10^tm1a/Y mice (Figure 2B, middle and bottom, Table 1). This extra pair of ribs linking to the sternum transforms the T8 vertebrae into an anterior T7-like phenotype (Figure 2—figure supplement 1, Table 1).

A majority of the Naa10^-/Y and Naa10^-/- mice also had four instead of the usual three sternebrae, which were sometimes fused (Table 1). Cervical vertebrae fusion was also demonstrated in Naa10^-/Y mice, particularly involving C1 and C2, suggesting possible anteriorization of C2 into a C1-like phenotype (Figure 2—figure supplement 1E, F, Supplementary file 1c). The number of lumbar vertebrae remained the same, thus suggesting an anterior transformation of the first sacral vertebra to a lumbar-like phenotype. These combined observations suggest possible anterior transformations in the Naa10 mutant skeletal phenotype, with an anteriorization of C2, a T8 transformation to a T7-like phenotype with ribs connecting to the sternum, an extra pair of ribs on L1 likely due to an L1 transformation to a T13-like phenotype, and an anterior transformation of the first sacral vertebra to a lumbar-like phenotype with loss of fusion to the sacral wings.

Out of 32 Naa10^-/Y that survived past the third day of life and which were then examined longitudinally, about 60% survived past 200 days of life (~7 months) (Figure 2—figure supplement 2), with some of these then developing hydronephrosis (Figure 2C, middle). They had some hollowed space in the kidney, which had been filled with fluid and their ureter was thickened already at P3 stage of prenatal development in some Naa10^-/Y mice (Figure 2—figure supplement 2A). Commonly, hydronephrosis is caused by a blockage or obstruction in the urinary tract. We speculate that this swelling in Naa10 KO (Naa10^-/Y and Naa10^-/-) mice is likely caused by ureteral defects rather than the kidney itself. Moreover, some Naa10 KO mice displayed genital defects, such as seminal vesicle malformation and hydrometrocolpos, respectively (Figure 2C, bottom). Many Naa10^-/- female mice appeared to have decreased fecundity, although they were fertile upon the first mating, and this decrease in fecundity is possibly due to the development of hydrometrocolpos (Figure 2C, bottom), which might result from structural issues, like vaginal atresia or a retained vaginal septum, although this requires further investigation. Additionally, hydrocephaly became clinically apparent with a round-shaped head (Figure 2C, upper) in ~40% of the Naa10^-/Y mice that had survived past 3 days of life (Figure 2—figure supplement 2C). CT scanning of some of these mice confirmed hydrocephaly as the primary cause of their rapid deteriorating condition, usually within the first three months of life (Figure 2—figure supplement 2B, C). CT scanning did not reveal any obstructive lesions (such as a tumor) in any of the ventricles that could account for the hydrocephaly. Taken together, these results indicate that Naa10 contributes to overall development and is particularly important for viability.

Litter sizes and offspring from other matings were also investigated, as shown in Supplementary file 1d. Matings were setup between Naa10^-/- females and C57bl6J WT (Naa10^+/Y) males, involving 11 mating pairs with seven unique females and seven unique males. Of a total of 127 pups that were born, 37 died in the first day of life and were degraded and/or cannibalized prior to any tail sample being retrieved, thus not being genotyped. This was a relatively high death rate in the first 24 hr of life (29%), more so than with the other matings, except for the one between Naa10^-/- females and Naa10^-/Y males (Supplementary file 1d). However, this is substantially less than the death rate of 90% (46/51) reported for the same mating in the Lee et al., 2017 paper, and we currently do not have an explanation for this discrepancy. Of the remaining 90 pups that could be genotyped, 59 of these were Naa10^+/- females and 31 were Naa10^-/Y males. 7 of the 59 Naa10^+/- females and 2 of the 31 Naa10^-/Y males died in the first three days of life (for a total death rate in the first three days for all born pups of 46/127, or 36%), and after this time, none of the remaining Naa10^+/- females died in the first 10 weeks of life (52/59, or 88% overall survival), whereas 10 of the remaining 29 Naa10^-/Y males developed hydrocephaly and died in the first 10 weeks of life, for an overall survival of (19/31, or 61%). The death rate for all pups of 36% in the first three days of life is similar to the rate of 42.4% seen with the mating of Naa10^-/- females with Naa10^-/Y males (Supplementary file 1d), whereas this rate is higher than that seen for Naa10^+/- females mated with Naa10^+/Y males (15.8%) or with Naa10^-/Y males (13.6%).

Prior experiments showed reduced in vivo protein amino-terminal acetylation of a few putative targets in patient cells (Myklebust et al., 2015). Reduced Nt-acetylomes were also observed in the Naa10 mutant yeast models (Van Damme et al., 2014). Given these prior reports, we hypothesized that pleiotropic phenotypes in Naa10-deficient mice are due to a decrease in global N-terminal acetylation. To test our hypothesis, integrated N-terminal peptide enrichment method (iNrich) (Ju et al., 2020) was used to analyze the level of protein amino-terminal acetylation in mouse embryonic fibroblast (MEF) lysates of Naa10^+/Y and Naa10^-/Y. Since the samples are treated with deuterated acetic anhydride prior to MS, unacetylated N-terminal site appears with +3 Da mass shift in the MS spectrum of the corresponding acetylated N-terminal site (Van Damme et al., 2011a). The peak intensity ratios of acetyl/heavy acetyl pairs represent the degree of acetylation of the N-terminal site. We found 765 acetyl/heavy acetyl pairs of N-termini throughout five replicates of Naa10^+/Y and five replicates of Naa10^-/Y MEFs. Except for the sites detected only in either WT or mutant, 533 N-terminal sites could be compared (see tabs called ‘N-term’ and ‘Header Key’ in Supplementary file 2a, c). Approximately 98% (n = 522) of N-termini sites showed less than 10% variation in the degree of terminal acetylation, indicating that there is no major difference in amino-terminal acetylation between Naa10^-/Y and Naa10^+/Y MEFs (Figure 3A). A more stringent analysis was also conducted in which peptides had to be detected in all 10 samples (i.e., tabs marked ‘N-term detected in all samples’ and ‘Header Key’ in Supplementary file 2b, c), and this resulted in 152 N-termini sites, of which only 3 (Rpl27, PPia, and Histone H1.0) had a slightly greater than 10% difference in the degree of acetylation between Naa10^+/Y and Naa10^-/Y. Although this was not a significant result statistically (p=0.09), it is worth noting that peptidyl-prolyl cis–trans isomerase A (PPIA), having a 10.3% decrease in amino-terminal acetylation, was previously identified with decreased amino-terminal acetylation in patient-derived B cells and fibroblasts in boys with the S37P mutation in NAA10 (Myklebust et al., 2015), along with being decreased in siNatA knockdown HeLa cells (Arnesen et al., 2009). PPIA also had decreased amino-terminal acetylation in one sample from homozygous null NAA15^L314*/L314*-induced pluripotent stem cells (Ward et al., 2021).

Overall, given the very minor differences with amino-terminal acetylation, we measured the in vitro amino-terminal acetylation activity of NatA via immunoprecipitation of the large auxiliary subunit Naa15 from mouse tissues. This analysis showed normal expression of Naa15 in Naa10 KO liver tissue as in WT tissues (Figure 3B), and we isolated a physical complex composed of Naa15 and undefined partners that retains NatA activity from Naa10 KO tissues (Figure 3C). These data suggest that despite the loss of Naa10 in mice the NatA complex remains active, thus explaining the lack of major differences with amino-terminal acetylation.

Naa10 disruption is lethal in a variety of organisms, including Drosophila melanogaster (Wang et al., 2010), C. elegans (Chen et al., 2014), and Trypanosoma brucei (Ingram et al., 2000). Given the relatively mild phenotype and no reduction of the Nt-acetylome in Naa10 KO mice, we hypothesized that there might be a yet unidentified paralog of Naa10, which can compensate for loss of function in mice. A Blast search for genomic sequences with homology to Naa10 exposed several Naa10 pseudogenes on chromosomes 2, 3, 7, 12, 15, and 18. Additionally, Southern blot analysis from C57BL/6J DNA with Naa10 cDNA probe detected bands of the expected sizes on the X chromosome (Figure 3—figure supplement 1A, B), while other bands of unexpected sizes appeared on other chromosomes 2, 5, 15, and 18. The previously identified Naa10 paralog Naa11 is located on chromosome 5; however, this paralog is only expressed in testes (Pang et al., 2011). We found a predicted gene (Gm16286, UniProt: Q9CQX6) on chromosome 18, with high similarity to Naa10, which we name Naa12, and RiboSeq and mRNA traces of this region suggest possible transcription and translation of this gene (Figure 3—figure supplement 1C). The protein sequence of Naa12 is >80% identical to Naa10 and almost 90% identical with Naa11 (Figure 4—figure supplement 1C).

Quantitative PCR (q-PCR) analysis also confirmed the expression of this transcript in all tested tissues (Figure 4—figure supplement 1A), with the expression of Naa12 unchanged in the corresponding Naa10 KO tissues. We attempted to test for Naa12 expression in mouse tissues by developing an antibody specific for Naa12 by performing a sequence alignment of the two known mNaa10 isoforms, mNaa11 and mNaa12, and selecting a unique Naa12 peptide for immunization and antibody generation (Figure 4—figure supplement 1B). After generation and affinity purification, we validated the specificity and sensitivity of this Naa12 antibody with recombinant proteins purified from bacterial hosts (Figure 4—figure supplement 1C, D). However, multiple attempts to use this antibody to detect Naa12 in mouse tissues met with conflicting results, so that we were unable to consistently detect Naa12 even in WT liver, kidney, or brain tissue lysates, which could be due to a poor antibody and/or very low expression or post-translational modification of Naa12 in these tissues, thus making it difficult to detect. Furthermore, given that this antibody was raised against a peptide at the C-terminus of Naa12, such data could not be used anyway to completely exclude the possibility of truncated non-functional mini-protein expression, although the lack of any signal with RT-PCR (Figure 4—figure supplement 2) likely means that nonsense-mediated decay occurred. Presently, the rabbit polyclonal antibody is no longer recognizing any consistent protein bands in western blotting, so we have abandoned any further attempts to use this antibody.

To test whether Naa12 has a similar enzymatic activity as Naa10, we performed a radioactive-based acetyltransferase assay using synthetic peptides (Figure 4A). Since monomeric Naa10 preferentially acetylates N-termini with acidic side chains (Foyn et al., 2013; Van Damme et al., 2011b; Liszczak et al., 2013), we used peptides representing the N-termini of γ-actin (starting DDDIA-) and γ-actin (starting EEEIA-), which are two known Naa10 in vitro substrates. Additionally, we used a peptide starting with SESSSKS-, representing an in vitro NatA complex substrate high-mobility group protein A1. As expected for the monomeric proteins, we could not detect any activity towards the SESSSKS substrate. Importantly, both Naa10 and Naa12 significantly Nt-acetylated the acidic N-terminal peptides, demonstrating the intrinsic capacity of Naa12 to catalyze amino-terminal acetylation (Figure 4A).

Across species, Naa10 is bound to its auxiliary subunit, Naa15, which links the catalytic subunit to the ribosome to facilitate co-translational amino-terminal acetylation of proteins as they emerge from the exit tunnel (Mullen et al., 1989; Sugiura et al., 2003; Park and Szostak, 1992; Gautschi et al., 2003; Magin et al., 2017; Varland and Arnesen, 2018). Due to its high sequence similarity (Figure 4—figure supplement 1B), we suspected that Naa12 may also interact with Naa15. To test this hypothesis, we performed co-immunoprecipitation assays in HEK 293 cells. Apart from Naa10 (isoform 1, Naa10²³⁵) and Naa12, we also included the second isoform of mNaa10, mNaa10²²⁵ that has been described earlier (Arnesen et al., 2005; Park and Szostak, 1992; Kim et al., 2006) as well as Naa11. Both Naa10 isoforms as well as Naa11 and Naa12 co-precipitated with V5-Naa15 but not V5 alone, suggesting that all tested proteins could form a stable complex with Naa15 in mouse (Figure 4B). As we have previously purified the human NatA complex composed of truncated human Naa10 (residues 1–160) and full-length human Naa15 complexes that had been expressed in insect cells (Gottlieb and Marmorstein, 2018), we attempted to co-express a chimeric truncated mouse Naa12 (residues 1–160) with full-length human Naa15 complex in insect cells (human and mouse Naa15 are highly conserved with a sequence conservation of 98.2%). The complex was purified by a combination of affinity, ion exchange, and size-exclusion chromatography, and size-exclusion fractions harboring a clearly detectable band of Naa15 and a lighter band for Naa12, as determined by silver staining, were analyzed for activity towards a SESSSKS- peptide (Figure 4C, Figure 4—figure supplement 1E, F). This analysis revealed that peak fractions containing the Naa12-Naa15 complex harbored detectable amino-terminal acetylation activity towards the SESSSKS- peptide (Figure 4C, Figure 4—figure supplement 1F), thus demonstrating catalytic activity of a NatA complex with mouse Naa12.

In a mass spectrometry analysis of a similar setup to that shown in Figure 3B, NAA15 immunoprecipitates from WT or Naa10-KO mouse livers were analyzed by mass spectrometry. We found five distinct peptides derived from Naa12 (Table 2 and Supplementary file 2d). Three of these derive from the same part of the peptide sequence, RDLSQMADELRR, and all of these three peptides had one or two missed trypsin cleavages (DLSQMADELRR, RDLSQMADELR, and RDLSQMADELRR). The other two peptides, AMIENFSAK and ENQGSTLPGSEEASQQENLAGGDSGSDGK, are not the results of missed cleavages. None of these peptides are found in other sequences in the mouse genome and thus unambiguously identify Naa12 in our experiments. They have higher intensities in Naa15 IPs compared to Ctrl IPs, indicating that Naa12 is selectively enriched by Naa15 IP. Some peptides are additionally assigned to the Naa10/Naa11/Naa12 protein group as a large part of their sequences are identical. As expected, no unique Naa10 peptides are identified in the IPs from Naa10-KO mice. 12 peptides were ambiguously assigned to Naa12 or to major urinary proteins (Mup9, Mup8, Mup1, Mup17, Mup5, or Mup2), but these are as likely to be derived from Mups as from Naa12, as they have comparable intensities between Ctrl and Naa15 IPs.

To investigate whether Naa12 can rescue the loss of the function of Naa10 in vivo, Naa12 KO mice were generated using CRISPR technology (Singh et al., 2015). One 95-base pair deletion Δ131–225 in Naa12 was characterized in depth (Figure 5A). This mutation introduces a frameshift, leading to a termination codon at amino acid 67, which should either result in complete KO of the protein or, at best, the expression of a truncated mini-protein that would be far shorter than the usual 220 amino acid Naa12. We confirmed the deletion by PCR with genomic DNA (Figure 5B). QPCR further showed deletion of Naa12 in the tested tissues of Naa12 KO mice (Figure 5C); however, it seemed that Naa12 might be slightly expressed in testis. Due to the high similarity between Naa11 and Naa12, the expression shown in Naa12 KO testis could actually be Naa11 rather than Naa12, and this was confirmed by RT-PCR showing definite deletion (Figure 4—figure supplement 2A).

Paralogs are homologous genes that originate from the intragenomic duplication of an ancestral gene. Homologs that play a compensatory role can sometimes show similar phenotypes to each other when one of them is deficient (Peng, 2019), whereas other homologs might only offer partial compensation when the primary gene is more widely expressed or has higher activity levels. We analyzed Naa12 KO mice to see if they produced similar developmental defects to those in Naa10 KO mice. KO mice for this gene were viable (Supplementary file 1e). Although there was initially a question of decreased fertility for the male mice, larger numbers of matings and litters did not bear this out (Supplementary file 1d), and necropsy and inspection of testes and seminal vesicles under a stereomicroscope did not reveal any macroscopic differences. Furthermore, the phenotypes (piebaldism and bilateral supernumerary ribs, Figure 2B) observed in Naa10 KO mice with complete penetrance were not present in Naa12 KO mice (Figure 4—figure supplement 2B). Overall, there were not any obvious phenotypes in these mice.

Matings between Naa10^+/- Naa12^+/+ female mice and either Naa10^+/y Naa12^+/- or Naa10^+/y Naa12^-/- males produced zero male Naa10^-/y Naa12^+/- progeny, while also suggesting that compound heterozygous (Naa10^+/- Naa12) female mice are produced at a rate much less than predicted by Mendelian ratios (Supplementary file 1f, g). Matings between surviving compound heterozygous (Naa10^+/- Naa12^+/-) females and Naa10^+/Y Naa12^+/- males demonstrate that no live births occurred for Naa10 Naa12 double-knockout (DKO) males (Naa10^-/Y Naa12^-/-) (Figure 6). In addition, the average litter size was small when compared to the control (WT × WT) matings, suggesting embryonic lethality (Table 3). In order to determine whether lethality occurs during the embryonic stage, we genotyped E18.5 litters – just before birth. Consistent with our previous observations, we could not obtain any Naa10^-/Y Naa12^-/- embryos, and many embryos could not be genotyped because they were already in the midst of resorption (n = 23) (Figure 6). We checked an even earlier stage at E10.5 and also found zero Naa10^-/Y Naa12^-/- embryos, and also with far fewer resorptions at this stage (N = 3). Interestingly, we did observe Naa10^-/Y Naa12^+/- embryos where two of them displayed delayed developmental stage (appearing younger than E10.5) and another two embryos were lysed and had already begun degenerating (but despite this, we could at least genotype these embryos). This helps explain why only one Naa10^-/Y Naa12^+/- embryo was observed at E18.5. Furthermore, Naa10^+/- Naa12^-/- female embryos were also lysed/degenerating at E10.5 and were not observed from that day onward. Matings between compound heterozygous females and Naa10^+/Y Naa12^-/- males also did not yield any Naa10^-/Y Naa12^-/- male mice at any embryonic stage examined, and only a couple of Naa10^+/- Naa12^-/- female mice at early stages of development (Figure 6—figure supplement 1), and the litter sizes were even smaller, suggesting increased embryonic lethality (Table 3). Consistent with this, we noted many resorptions at E12.5 and E18.5 that could not be genotyped. The number of living postnatal compound heterozygous female mice was also considerably lower than the predicted Mendelian ratios (Figure 6, Figure 6—figure supplement 1) and the surviving Naa10^+/- Naa12^+/- females were smaller in size than littermate controls (Figure 7D).

Due to the severe embryonic lethality observed in the Naa10 Naa12 DKO male mice and the Naa10^+/- Naa12^-/- female mice, which was not seen in each single KO (Naa10 KO or Naa12 KO), it seems likely that, without compensation by Naa12, amino-terminal acetylation is disrupted in Naa10 Naa12 DKO mice. Together, these data support the compensatory role of Naa12 in vivo.

The discrepancies we noted between the observed offspring genotype distributions and the expected Mendelian frequencies prompted us to examine the results from four matings (Supplementary file 1h–m) with the goal of understanding the effects of combined Naa10 and Naa12 mutations on embryonic and postnatal mortality. We created mathematical models to predict the observed genotype distribution at each age based on successive incorporation of assumptions of the lethality of specific offspring genotypes. Embryonic genotype data was obtained from two matings for which embryonic genotype data were obtained (Supplementary file 1h, i). Those matings were (1) Naa10^+/Y; Naa12^+/- males crossed with Naa10^+/-; Naa12^+/- females (Figure 6, Supplementary file 1h) and (2) Naa10^+/Y; Naa12^-/- males crossed with Naa10^+/-; Naa12^+/- females (Figure 6—figure supplement 1, Supplementary file 1i). The genotype numbering shown in Supplementary file 1h was used throughout this analysis, and the corresponding genotypes for all other crosses are aligned to have the same numbers. Each model described below adjusted the expected observed genotype frequencies at each age to account for loss of embryos or pups due to the predicted lethal effects of one or more genotypes by the method described in Materials and methods. Three stages of models (B–D) were compared with the expected Mendelian distribution (model A).

Model B assumed that the double KO male genotype 12 (Naa10^-/Y; Naa12^-/-) is lethal from very early in development based on the observation that this genotype was not found in any embryos or pups out of 483 obtained genotypes from all litters. Specifically, 0 out of an expected 7.9 were detected at E10.5 or earlier, 0 out of an expected 14.5 were detected at E18.5 or earlier, and 0 out of an expected 46.7 were detected by P3. Thus, the survival for genotype 12 was 0% for all ages examined.

Model C was developed from model B in two stages by incorporating separately observations that the male genotype 11 (Naa10^-/Y; Naa12^+/-) and the female genotype 6 (Naa10^+/-; Naa12^-/-) were lethal during mid to late fetal development. Based on the Mendelian model, 5 of 9.8 (51%) expected genotype 11 were detected by E10.5 but only 1 of expected 8.6 (11.6%) were identified on E12.5 or E18.5 and none were detected at P3. The five embryos that were present at E10.5 were noted to be lysed and/or developmentally delayed; the single E18.5 genotype 11 embryo was not observed to be abnormal. Based on the Mendelian model for genotype 6, 1 of 2.6 expected E8.5 embryos and 3 of 5.3 expected E10.5 embryos were identified. All three E10.5 embryos were identified as lysed. Genotype 6 was not identified after age E10.5. Cumulatively, 4 of 7.9 expected embryos detected by E10.5 and 0 of 38.8 expected embryos/pups thereafter.

Model D incorporated the assumptions of models B and C and added adjustments to the survival rates of genotype 5 (Naa10^+/-; Naa12^+/-) and genotype 10 (Naa10^-/Y; Naa12^+/+) based on the observations that these genotypes were underrepresented at late fetal ages or early postpartum. Genotype 5 was overrepresented during embryogenesis (31 identified but only 18.4 expected for all embryonic ages) but was underrepresented at P3 (17 of 42 expected) based on the expected Mendelian frequencies. A better analysis was achieved by comparing the observed genotype frequencies with those predicted by model C because the expected distributions are significantly affected by the lethal effects of the three genotypes considered in that model. In that case, the genotype overrepresentation during embryogenesis is somewhat less (31 identified but only 22.1 expected) but the underrepresentation at P3 is significantly increased (17 identified of 62 expected, or 27%). Using a model (D₃) that incorporated adjusted survival rates for genotypes 12, 11, 6, and 5, we found that genotype 10 remained underrepresented in the observed postnatal offspring counts. The subsequent model (D₄) incorporated adjustments to genotype 10 survival rates at E18.5 and P3 to account for this and then was slightly refined by adjusting other genotype survival rates to maximize the fit of all genotypes at all ages. The survival values for model D₄ are shown in Table 4. A comparison of the observed offspring numbers with those predicted by the Mendelian distribution and model D₄is shown in Figure 6—figure supplements 2–5.

The observations of reduced survival for selected Naa10/Naa12 mutants suggests that Naa10 is the more dominant function (e.g., is able to provide Naa12 functions more successfully than Naa12 can provide Naa10 functions) but that two copies of Naa10 are required to replace complete loss of Naa12 in females, possibly due to X-linked inactivation of Naa10 during development. The stochastic nature of X-linked inactivation in time and space may make Naa10 functionality somewhat unpredictable during development in a background having a mixture of Naa10 and Naa12 mutations.

To determine whether Naa10 and Naa12 are essential for viability and development, we examined the survival, weights, and growth rates of 688 Naa10 and Naa12 KO and WT mice. The genotypes of mice examined are listed in Supplementary file 1n. To avoid potential survival biases, only weights taken during the first 180 days were included. Growth curves are shown in Figure 7. Age and age-squared (the quadratic term) are both entered in the analyses; the quadratic term shows the degree to which the effect of age itself changes with age.

Supplementary file 1o shows the results in which the weight of Naa10 mice in grams is regressed upon age, Naa10 KO status, and their interaction. Unsurprisingly, age predicts weight for males and females strongly, with growth slowing with age (first column). Though a strong negative effect of the KO is seen in for both males and females (second column), when both age and KO status are modeled together (third column), this effect all but disappears in females. Moreover, in females there is no interaction of KO status with age (fourth column), suggesting that the Naa10 KO status itself has no significant effect on the growth rate in females. For males, however, the main effect of the KO remains when age is included in the model (third column) and the interaction is significant (fourth column), indicating that the Naa10 KO both reduces weight of males overall and lowers the rate of growth.

Results of analyses of mixed-genetic background Naa10/Naa12 mice are shown in Supplementary file 1p. Effects of age and KOs on weight comprise the upper portion of the table, while the lower portion shows their effect on the rate of weight gain. Among females, a significant reduction of weight (above, second column) and in the rate of growth (below, first column) is seen among mice heterozygous for the Naa10 KO. There were no homozygous Naa10 KO female mixed-breed mice available to analyze as the matings were not setup to yield any such mice (so breeding patterns, not mortality in utero, are the reason for this absence). No significant effect on growth rate is seen for heterozygous or homozygous Naa12 KO (above, third column) or for their interactions with age (below, second column), and only the effects of the heterozygous Naa10 KO and its interaction with age are seen in the full model (below, third column). Thus, the Naa12 KO, whether heterozygous or homozygous, does not appear to reduce the weight or growth rate of females, while a heterozygous Naa10 KO is sufficient to reduce both weight and growth rate. Interestingly, when modeled together, both the Naa10 and the Naa12 KOs significantly reduced weight (above, fourth column) and the interaction of the Naa10 and the Naa12 heterozygous KOs significantly reduced weight (above, fifth column). As no female mice were both KO for Naa10 and homozygous KO for Naa12, the effect of the interaction of those two factors could not be determined. The triple interaction of heterozygous Naa10 KO, Naa12 KO, and age was weakly significant, suggesting that the presence of both KOs affects growth rate above and beyond the effects of each KO independently (below, fourth column). No males with KOs of both Naa10 and Naa12 were born, so no test of their interaction was possible. An effect was seen for the Naa10 KO on weight when modeled with age and age² (second column), and the significant interaction of the Naa10 KO with age and age² (third column) shows that the Naa10 KO in males reduces the growth rate. As with females, no significant effect of a Naa12 KO, whether heterozygous or homozygous, was seen in males, nor is there a significant interaction with age (fourth column). When the interactions of age with both Naa10 and Naa12 KO status are entered in one model, Naa10 alone is seen to reduce growth rates (fifth column).

All experiments were performed in accordance with guidelines of International Animal Care and Use Committee (IACUC) of Ewha Womans University (protocol #18-012), Cold Spring Harbor Laboratory (CSHL) protocol #579961-18, and Institute for Basic Research in Developmental Disabilities (IBR) (protocol #456). At CSHL and IBR, any matings that required genotyping were screened on a daily basis by animal husbandry staff, with notation of how many newborn pups were present each morning, but with paw tattoo and tail genotyping not being performed until day 3 of life, so as to not disturb the litters and thus to not increase the risk for maternal rejection of the litter. The stock of C57BL/6J was replenished annually from Jackson Laboratory so as to avoid genetic drift from the JAX inbred line.

The Naa10 KO mice were generated as previously described (Yoon et al., 2014). Naa10^tm1a [B6;129P2-Ard1^{tm1a(Eucomm)Gto}/J] (Naa10^tm1a) mice, used for Naa10 reporter mouse, were generated using standard method based on a standard gene-targeting in E14 embryonic stem (ES) cells (129/Sv) by using a targeting vector from EUCOMM. Correctly targeted ES clones were used for blastocyst microinjection and generation of chimeric mice. Chimeric mice were crossed to C57BL/6J mice, and then the progeny were backcrossed to C57BL/6J for more than 10 generations. The Naa10-deficient mice used in the weight analyses were derived from mice backcrossed eight times to a C57BL/6J inbred genetic background, and this was confirmed with genome scanning at the Jackson Laboratory, showing heterozygosity for only one marker for 129S1/SvImJ out of 290 autosomal markers tested, thus giving a percentage of C57BL/6J of 99.66%.

The mice were made using standard methods by microinjection of CRISPR reagent mix into zygotes obtained from the mating of B6D2F1 females (i.e., 50% C57BL/6J, 50% DBA/2J [D2]) females to inbred C57BL/6J males. The guide RNA was produced and validated from Sigma using a Cel1-nuclease assay, and the most active guide was selected, which was Naa12_0_125 (C9587), with a target sequence of GAGCGTTTCACAGCCAGCG and including the targeting cr-RNA sequence and the tracrRNA portion. The indels were transmitted by breeding again to inbred C57BL/6J males, and the resulting progeny were interbred on a mixed genetic background of approximately 12.5% DBA/2J (D2)/87.5% C57BL/6J, for use in the reported experiments, including the weight analyses. Progeny from these mice have been backcrossed to C57BL/6J for more than 10 generations, with no discernible new phenotypes emerging. Genomic DNA was isolated from paw and tail. DNA was screened for mutations using PCR and Surveyor assay (Qiu et al., 2004), followed by Sanger sequencing of selected clones and the use of CRISP-ID (Dehairs et al., 2016) to identify putative deletions.

The primers used for Naa10 KO and Naa10^tm1a genotyping were Naa10-F: 5′-cctcacgtaatgctctgcaa-3′, Naa10-neo-F: 5′-acgcgtcaccttaat-atgcg-3′, Naa10-R: 5′-tgaaagttgagggtgttgga-3′, Naa10^tm1a-F: 5′-gcacactctctgaattggac-3′, Naa10^tm1a-neo-F: 5′-ggccgcttttctggattcat-3′, and Naa10^tm1a-R: 5′-gcaggggaataaggcattgg-3′. The primers used for Naa12 KO were Naa12 Surveyor F: 5′-gctccacctcgctaacctgg-3′, Naa12 Surveyor R: 5′-gccagatgacctgatgaacatgc-3′ and HEX-Naa12 Surveyor F: 5′-gctccacctcgctaacctgg-3′.

The following antibodies were used: rabbit anti-Naa10 (Abcam #ab155687), rabbit anti-Naa10 (Protein Tech #14803-1-AP), rabbit monoclonal anti-NAA10 (Cell Signaling, #13357), goat anti-Naa10 (Santa Cruz, #sc-33256), rabbit anti-Naa10 (Santa Cruz, #sc-33820), rabbit anti-Naa11 (Novus Biologicals; #NBP1-90853), mouse anti-Naa15/NARG1 (Abcam; #ab60065), rabbit polyclonal anti-NAA15 (Arnesen et al., 2005), rabbit anti-Naa50 (LifeSpan BioSciences; #LS-C81324-100), rabbit anti-FLAG (Sigma; #F7425), mouse anti-GAPDH (Abcam; #ab9484), goat anti-actin (Santa Cruz, #1615), mouse anti-GST (GenScript; #A00865), and mouse anti-V5 (Life Technologies; #R960-25). The antibody against the potential mNaa10 paralog mNaa12 (Gm16286, UniProt: Q9CQX6) was raised in rabbits after immunization with a synthetic peptide of the Naa12 C-terminus (aa191-205: QENLAGGDSGSDGKD-C) conjugated to OVA by PrimmBiotech.

After the skin and internal organs were removed, embryos were fixed in 95% ethanol (EtOH) for 4 hr, then in 100% acetone for overnight. Embryos were stained with 0.03% Alcian Blue 8GX in ethanol/acetic acid (4:1 v/v) for overnight and kept in 1% KOH for 2 days until they became clearly visible, followed by staining with 0.05% Alizarin Red in 1% KOH for 4 hr. After washing with 100% glycerol/1% KOH (1:1 v/v), skeletons were kept in 100% glycerol.

Timed matings were performed either by using the presence of a vaginal plug to assess fertilization. The morning vaginal plug was designated E0.5. Pregnant mice were sacrificed at several time points after conception. The embryos were isolated in ice-cold PBS with 1% FBS and washed three times in ice-cold PBS. Embryos were imaged using a Zeiss Axiozoom V16 with Zen software and merged 50 slides between Z-stack intervals.

Isolated E10.5 embryos were incubated in fixation solution (4% paraformaldehyde) at 4°C for 25 min. Samples were washed in ice-cold PBS and then incubated in permeabilization solution (PBS containing 0.01% Na deoxycholate, 0.02% Nonidet-P40, 2 mM MgCl₂) for 20 min at 4°C. Subsequently, samples were incubated in β-gal staining solution (PBS containing 1 mg/mL X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 0.02% Nonidet-P40, 2 mM MgCl₂) at 37°C overnight. Following β-gal staining, samples were washed with PBS and incubated in fixation solution at 4°C for storage.

Isolated kidney tissues at E18.5 and P3 were fixed with 4% paraformaldehyde at 4°C for overnight and embedded in paraffin. Samples were sectioned at 8 μm thick and stained with hematoxylin (MHS80, Sigma) and eosin (HT110116, Sigma) for morphology.

Full-length mouse Naa10 and Naa12 (Gm16286, UniProt: Q9CQX6) expression vectors were separately constructed using a pMAL-c5x vector. In both cases, the catalytic subunit contained an N-terminal uncleavable MBP-tag. Bacterial expression vectors of mNATs were cloned from cDNA generated from mouse liver or testes. mRNA was isolated using the Oligotex direct mRNA kit (Qiagen) according to the manufacturer's recommendations. 1 µg RNA was reverse transcribed with Superscript IV reverse transcriptase (Thermo Fisher) and Oligo dT(18) primer. The PCR product was digested and cloned into BamHI restriction sites of pGEX-4T1 (GE Healthcare), pMAL-p5X and p3xFLAG-CMV10 (Sigma-Aldrich) using standard techniques. All constructs were sequenced to validate correct insert and orientation.

cDNA was amplified using the primers CCG GGA TCC ATG AAC ATC CGC AAT and CTG GGA TCC CTA GGA GGC AGA GTC AGA for mNaa10 variants, CCG GGA TCC ATG AAC ATC CGC AAT GC and CTG GGA TCC CTA GGA GAT GGA ATC CAA GTC for mNaa11, CCG GGA TCC ATG AAC ATC CGC CGG and CTG GGA TCC CTA GGA GGC GGA CCC TAG for mNaa12.

To determine the specificity of the mNaa12 antibody, a peptide competition assay was performed using the same peptide as used for immunization (aa 191–205: QENLAGGDSGSDGKD-C). 100 µg antibody were bound to 50 mg peptide-coupled CNBr-Sepharose (10 mg peptide/g Sepharose) in PBS + 0.2% Triton X-100 for 1 hr at 4° C on an orbital shaker. The beads were pelleted by centrifugation at 2.700 × g for 3 min at 4°C and 250 µL of the antibody-depleted supernatant diluted in 5 mL TST for detection (1:100 final antibody dilution). Western blots of mouse lysates were probed with the depleted antibody or untreated antibody as control (1:100 dilution in TST).

HEK293 cells were purchased from ATCC, authenticated via STR profiling, and confirmed mycoplasma free.

Protein-protein interaction studies were performed in HEK293 cells. Briefly, 8 × 10⁵ cells were seeded per well in 6-well plates. After 24 hr, cells were co-transfected with pcDNA3.1/V5-His-mNaa15 and p3xFLAG-CMV10-Naa10²³⁵ (isoform 1), -Naa10²²⁵ (isoform 2), -Naa11, or -Naa12 or the corresponding empty vectors. Cells were lysed after 48 hr in 200 µL PBS-X per well and cellular debris pelleted at 20.800 × g for 10 min at 4°C. 350 µL of the generated lysate was incubated with 1 µg anti-V5 antibody for 1 hr at 4°C, followed by a 30 min incubation with 30 µL protein-A Sepharose (Sigma-Aldrich). Protein complexes were washed three times by centrifugation (2.700 × g, 2 min) and eluted in 30 µL 2×SDS sample buffer.

Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Protran 0.2 µM NC) by immunoblotting. The membrane was blocked in 5% non-fat dry milk and incubated overnight with rabbit polyclonal anti-NAA15 (Arnesen et al., 2005) (1:2000, BioGenes) and rabbit monoclonal anti-NAA10 (anti-ARD1A, 1:1000, Cell Signaling, #13357) diluted in 1× PBS containing 1% non-fat dry milk and 0.1% Tween. The immunoblots were washed and incubated for 1 hr at room temperature (RT) with HRP-linked secondary antibody donkey anti-rabbit IgG (GE Healthcare, NA934). The HRP-signal was detected using SuperSignalTM West Pico PLUS Chemiluminescent Substrate Kit (Thermo Scientific) and ChemiDocTM XRS+ system (Bio-Rad) and visualized by ImageLab Software (Bio-Rad).

For immunoprecipitation of Naa15, 90–120 mg liver tissue from a WT- and Naa10 KO mouse was lysed in 500 µL IPH lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM) NaCl, 5 mM EDTA, 0.5% NP-40, 1× complete EDTA-free protease inhibitor cocktail (Roche) using Kontes Pellet Pestle Motor and incubated on ice for 40 min. Cell debris was pelleted by centrifugation (17,000 × g, 4°C, 10 min) and the supernatants transferred to new Eppendorf tubes. The protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific) and the tissue lysates were subsequently diluted with IPH lysis buffer to an equal protein concentration of 25 µg/µL. The WT- and Naa10 KO tissue lysates were then divided in two, whereof one half was mixed with 15 µg of anti-Naa15 antibody and the other half with 15 µg of anti-V5 antibody as a negative control. The mixtures were incubated at 4°C for 3 hr on a rotator. Afterwards, 180 µL of Protein A/G magnetic beads (Millipore) pre-washed in IPH lysis buffer was added to each sample and incubated overnight. Then, the magnetic beads were washed three times in IPH lysis buffer and two times in 1× acetylation buffer (100 mM Tris-HCl pH 8.5, 2 mM EDTA, 20% glycerol) prior to being resuspended in 90 µL of 2× acetylation buffer and used in a [¹⁴C]-Ac-CoA-based acetylation assay.

Three positive replicates were prepared for each IP sample containing 10 µL IP beads, 200 µM synthetic oligopeptide SESS₂₄ (BioGenes), 100 µM [¹⁴C]-Ac-CoA (Perkin-Elmer), and dH₂O to a final volume of 25 µL. In addition, two replicates for each IP sample were prepared without synthetic oligopeptide as negative controls. The samples were incubated at 37°C for 45 min in a thermomixer with shaking at 1400 rpm. Finally, the magnetic beads were isolated and 23 µL of the supernatant transferred to P81 phosphocellulose filter disks (Millipore). The filter disks were washed three times for 5 min in 10 mM HEPES buffer (pH 7.4) and air dried. To determine the amount of incorporated [¹⁴C]-Ac, the filter disks were added to 5 mL Ultima Gold F scintillation mixture (Perkin-Elmer) and analyzed by a Perkin-Elmer TriCarb 2900TR Liquid Scintillation Analyzer.

Immunoprecipitation of Naa15 from a WT- and Naa10 KO mouse was performed as described above. Bound proteins were eluted from the magnetic beads using 60 µL of elution buffer (2% SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT) and heated for 5 min at 95°C. The eluates were processed for LC-MS/MS analysis using filter-aided sample preparation (FASP) method (Wiśniewski et al., 2009). The eluted protein mixtures were mixed with UA buffer (8 M urea, 100 Mm Tris-HCl pH 8.0) and centrifuged through Microcon 30 kDa MWCO filters followed by Cys-alkylation with 50 mM iodoacetamide dissolved in UA buffer. Afterwards, the buffer was exchanged with 50 mM ammonium bicarbonate through sequential centrifugation, proteins were trypsinized (Sequencing Grade Modified Trypsin, Promega), and digested peptides were collected by centrifugation. Peptides were acidified using 5% formic acid and desalted using C18-stagetips according to protocol (Rappsilber et al., 2007). Briefly, 40 µg peptides from each sample were loaded onto C18-stagetips pre-conditioned with buffer A (1% formic acid). The C18-stagetips were then washed with buffer A, before peptides were eluted with buffer B (80% acetonitrile [ACN], 1% formic acid). The final eluate was concentrated by Speedvac to evaporate ACN and diluted to desired volume with 5% formic acid.

1 µg of the peptide samples were injected into an Ultimate 3000 RSLC system (Thermo Scientific) connected to a Q-Exactive HF mass spectrometer (Thermo Scientific) equipped with EASY-spray nano-electrospray ion source (Thermo Scientific). Trapping and desalting was performed with 0.1% TFA (flow rate 5 µL/min, 5 min) on a pre-column (Acclaim PepMap 100, 2 cm × 75 µm ID nanoViper column, 3 µm C18 beads). Peptides were separated on an analytical column (PepMap RSLC, 50 cm × 75 µm i.d. EASY-spray column, 2 µm C18 beads) during a biphasic ACN gradient with a flow rate of 200 nL/min. Solvent A (0.1% FA [vol/vol] in water) and B (100% ACN) were used for the following gradient composition: 5% B for 5 min, 5–8% B for 0.5 min, 8–24% B for 109.5 min, 24–35% B for 25 min and 35–80% B for 15 min, 80% B for 15 min, and conditioning with 5% B for 20 min. The mass spectrometer was operated in data-dependent mode to automatically switch between full-scan MS and MS/MS acquisition. MS spectra (m/z 375–1500) were acquired with a resolution of 120,000 at m/z 200, automatic gain control (AGC) target of 3 × 10⁶, and maximum injection time (IT) of 100 ms. The 12 most intense peptides above an intensity threshold (50,000 counts, charge states 2–5) were sequentially isolated to an AGC target of 1 × 10⁵ and maximum IT of 100 ms and isolation width maintained at 1.6 m/z, before fragmentation at a normalized collision energy of 28%. Fragments were detected in the orbitrap at a resolution of 15,000 at m/z 200, with first mass fixed at m/z 100. Dynamic exclusion was utilized with an exclusion time of 25 s and ‘exclude isotopes’ enabled. Lock-mass internal calibration (m/z 445.12003) was used. Raw files were processed with MaxQuant v. 1.6.17.0 (Cox and Mann, 2008) and searched against a database of Swiss-Prot annotated mouse protein sequences (retrieved 22.06.2018) in which the NAA12 sequence was added manually, and with a reverse decoy database. MaxQuant was run with default settings. Peptide and protein identifications were filtered to a 1% false discovery rate (FDR). Minimum peptide length was set to 7. Modifications included in protein quantification were oxidation (M), Nt-acetylation, acetylation (K), and phosphorylation (STY). Other parameters: match between runs – true, matching time window – 0.7 min, alignment time window – 20 min, find dependent peptides – true, mass bin size – 0.0065. Protein and peptide intensities were quantified by label-free quantification (LFQ) (Cox et al., 2014). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD026684.

CT scans were acquired on a Nanoscan PET/CT scanner from Mediso using Nucline v2.01 software. All mice were kept sedated under isoflurane anesthesia for the duration of the scan. Scans were acquired with an X-ray tube energy and current of 70 kVp and 280 uA, respectively. 720 projections were acquired per rotation, for three rotations, with a scan time of approximately 11 min, followed by reconstruction with a RamLak filter and voxel size 40 × 40 × 122 µm. For ex vivo analyses, mouse heads were fixed in 10% formalin buffered saline, followed by scanning and reconstruction with 1440 projections per revolution. Cranial volume was measured using VivoQuant software (v2.50patch2) using the spline tool to manually and accurately draw around the circumference of the cranium on multiple stepwise 2D slices.

iNrich assays were performed as described (Ju et al., 2020). MEFs were made from E13.5 embryos, using standard techniques, with DMEM media supplemented with 10% fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin. Cells were harvested by trypsinization, washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.4; Gibco), and resuspended in ice-cold lysis buffer containing 0.2 M EPPS (pH 8.0), 6 M guanidine, 10 mM TCEP (Thermo Fisher Scientific), and 40 mM 2-chloroacetamide (Sigma-Aldrich). After 10 min of incubation on 95°C, cells were lysed by ultrasonication by a BranSonic 400B. The proteins from the cell lysate were isolated by transferring supernatant after centrifugation at 12,000 g for 10 min at 4°C. The protein concentration of the collected supernatant was determined by bicinchoninic acid (BCA) protein assay. After enrichment of the N-terminal peptides, the peptide samples were analyzed by LC–MS/MS on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) without further fractionation. Mass spectrometry data were uploaded to PRIDE under project name: Naa10 mutant mouse N-terminome LC-MS, project accession: PXD026410. Data analysis used unpaired, equal variance algorithm for Student’s t-test.

70–120 mg tissues were lysed in 5 μL/mg tissue RIPA buffer (Sigma) with 1× Complete protease inhibitors and 1 U/μL Superase In RNase inhibitor (Thermo Scientific) using Fisherbrand Pellet Pestle Cordless Motor. Afterwards, homogenization debris was removed by centrifugation at 20.800 × g for 10 min at 4°C. Protein concentration was determined using APA assay (Cytoskeleton Inc) and 50 μg total protein were separated on SDS-PAGE followed by western blot. Membranes were stained with anti-Naa10, anti-Naa15, and anti-GAPDH antibodies (all Abcam).

For RNA purification, 30 μL clarified lysates were mixed with 70 μL RNase free water and RNA isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers recommendations, including on-column Dnase digest. 1 μg RNA was reverse transcribed using the TaqMan Reverse transcription kit and gene level detection performed using SYBR Green Master Mix (all Thermo Scientific). Relative expression was normalized to GAPDH and ACTB.

For the characterization of the mNaa12 antibody, tissue was lysed in 2 µL per mg tissue PBS-X (PBS + 0.2% [v/v] Triton X-100 + 1× Complete protease inhibitor cocktail). 10–200 µg lysate were subjected to SDS-PAGE and western blot.

The following primers pairs were used: mNaa10-Exon2/3 F: 5′-ctcttggccccagctttctt-3′ and mNaa10-Exon3/4 R: 5′- tcgtctgggtcctcttccat-3′, mNaa11-F: 5′-accccacaagcaaagacagtg-3′ and mNaa11-R: 5′-agcgatgctcaggaaatgctct-3′, mNaa12(Gm16286)-F: 5′-acgcgtatgctatgaagcga-3′ and mNaa12(Gm16286)-R: 5′-ccaggaagtgtgctaccctg-3′, mNaa15-F: 5′-gcagagcatggagaaaccct-3′ and mNaa15-R: 5′-tctcaaacctctgcgaacca-3′, mNaa50-F: 5′-taggatgccttgcaccttacc-3′ and mNaa50-R: 5′-gtcaatcgctgactcattgct-3′, mGAPDH-F: 5′-aggtcggtgtgaacggatttg-3′ and mGAPDH-R: 5′-tgtagaccatgtagttgaggtca-3′, mACTB-F: 5′-ggctgtattcccctccatcg-3′ and mACTB-R: 5′-ccagttggtaacaatgccatgt-3′.

All constructs were expressed in Rosetta (DE3)pLysS competent Escherichia coli cells. Cells were grown in LB-media to OD₆₀₀ 0.6–0.7 prior to inducing protein expression with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C for ~16 hr. All subsequent purification steps were carried out at 4°C. Cells were isolated by centrifugation and lysed in lysis buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, 10 mM β-mercaptoethanol (β-ME), 10 µg/mL phenylmethanesulfonylfluoride (PMSF), and DNase. The lysate was clarified by centrifugation and incubated with amylose agarose resin (New England Biolabs) for 1 hr before washing the resin with ≥100 column volumes of lysis buffer and then eluted with 10-column volumes of lysis buffer supplemented with 20 mM maltose. The resulting eluent was pooled and concentrated to ~10 mg/mL (30 kDa concentrator; Amicon Ultra, Millipore) such that 500 µL was loaded onto a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare). The gel filtration run was performed in sizing buffer containing 25 mM HEPES, pH 7.0, 200 mM NaCl, and 1 mM TCEP. After confirming the purity of the peak fractions at ~14 mL by denaturing SDS-PAGE (15% acrylamide), peak fractions were concentrated to 0.6 (6.1 µM) WT mouse Naa10 and 0.3 mg/mL (3.5 µM) WT mouse Naa12, as measured by UV₂₈₀ (Nanodrop 2000; Thermo Fisher Scientific), and stored at 4°C.

For recombinant mNaa12 and mNaa10 constructs, the assays were carried out in 40 mM HEPES, pH 7.5, 200 mM NaCl, where reactions were incubated with 150 nM of the gel-filtration purified WT mouse Naa10 or Naa12 in a 30 µL reaction volume containing each 250 µM substrate peptide and radiolabeled [¹⁴C]acetyl-CoA (4 mCi/mmol; PerkinElmer Life Sciences) for 12 min (Naa12) or 13 min (Naa10) at 25°C. Respective time points were selected to ensure detection of sufficient activity within the linear range as determined by a time-course experiment. The substrate peptides used in the assay correspond to the first seven amino acids of β-actin (DDDIAAL-), γ-actin (EEEIAAL-), or the in vivo NatA complex substrate high-mobility group protein A1 (SESSS-), along with C-terminal positively charged residues for capture to the anion exchange paper. Background control reactions were performed in the absence of enzyme or in the absence of substrate peptide to ensure that any possible signal due to chemical acetylation was negligible. Each reaction was performed in triplicate.

To quench the reaction, 20 µL of the reaction mixture was added to negatively charged P81 phosphocellulose squares (EMD Millipore), and the paper disks were immediately placed in wash buffer (10 mM HEPES, pH 7.5). The paper disks were washed three times, at 5 min per wash, to remove unreacted acetyl-CoA. The papers were then dried with acetone and added to 4 mL of scintillation fluid, and the signal was measured with a PerkinElmer Life Sciences Tri-Carb 2810 TR liquid scintillation analyzer. The counts per minute were converted to molar units using a standard curve of known [¹⁴C]acetyl-CoA concentrations in scintillation fluid.

Full peptide sequences:

For mNaa12-hNaa15, reactions were carried out similar to the monomeric mNaa12 and mNaa10, with the following exceptions: reactions were prepared by combining 21 µL of the respective fraction or sizing buffer with 5 µL of 10X buffer (500 mM HEPES, pH 7.5) to yield a buffer composed of 50 mM HEPES, pH 7.5, 140 mM NaCl, 0.7 mM TCEP, and 250 µM of each substrate upon reaction initiation. The reactions were allowed to incubate overnight at ambient temperatures (~25°C) and then quenched as described above. Control reactions were conducted in parallel as described above without conversion to molar units. Two technical replicates of the reactions were performed.

Significant differences (p<0.05) are indicated by asterisks. Weight analyses were performed using generalized estimating equations (GEEs) (Zeger and Liang, 1986), an extension of generalized linear models that adjusts for the effects of autocorrelation resulting from multiple measurements, and implemented within version 15.1 of Stata (StataCorp 2017).

Genotype distributions for several Naa10/Naa12 KO crosses were analyzed and models were created to estimate the number of the live (or at least intact) embryos or pups that are expected to be observed based on the assumptions and rules that follow. (1) Genotype survival rates are the fractional value, from 0 to 1 (or 0–100%), of the expected Mendelian fraction for that genotype in the cross being evaluated. (2) Genotype survival rates cannot exceed 1 (or 100%). (3) Genotype survival rates can decrease with age but not increase. (4) WT genotypes (Naa10^+/Y; Naa12^+/+ and Naa10^+/+; Naa12^+/+) are expected to have 100% survival at all ages because the models predict the number of embryos or pups relative to WT survival. Reductions in overall in litter sizes for crosses were estimated through other calculations. (5) The biological basis for a reduced survival rate assumes that loss of one or more copies of either Naa10 or Naa12 removes or reduces functions that are required for successful embryonic development or postnatal life. Reduced survival rates for non-WT genotypes were estimated based on differences (delta) between the expected number of embryos or pups based on the Mendelian proportion (or the current best model) and the observed number of embryos or pups. Separate comparisons were made using deltas for each specific age and for the cumulative numbers at each age. (6) Genotype frequencies for each model were calculated as described in the section below. (7) The fit between a model and the observed data was determined by calculating the relative standard deviation (SD) for the deltas across all genotypes, for example, the SD across genotypes divided by the number of animals observed (either age-specific or cumulative). Each model was evaluated at each age by minimizing the relative SD for all genotypes at that age and over all ages. The final model (D₄) was created by refining the assumptions for model D₃ in a sequential series of comparisons of survival rates for genotypes 12, 11, 6, 10, and 5 in that order.

The models described adjust the expected observed genotype frequencies at each age to account for loss of embryos or pups due to the presumed lethal effects of one or more genotypes. The models account directly for the effect of genotype-specific mortality by reducing the number (or frequency) observed for that genotype in the sample and thus increasing the expected proportion of other genotypes. This also indirectly implies a larger theoretical litter size at conception, which can be used to determine the theoretical litter sizes had there been no mortality in the affected genotypes. The predicted proportion for each genotype is calculated at each age as the genotype Mendelian frequency multiplied by the fractional genotype survival at that age divided by the expected total fractional survival (i.e., one minus the sum of all genotype fractional losses). The formula is

For all ‘n’ possible genotypes:Gx=Mx∗Sx/(1–[(1–S1)∗M1+(1–S2)∗M2+…+(1–Sn)∗Mn)]where

(1 – S_n) * M_n is the fractional reduction due to survival < 100% for genotype ‘n’, for example, when S_n = 1 (e.g., 100% survival), the loss is zero; when S_n = 0 (e.g., 0% survival), the loss is M_n or the entire Mendelian fraction.

Note that the sum of all G_x for all genotypes at any age is always equal to 1 (or 100%).