Actin N-terminal maturation scheme and the in vitro acetyltransferase activity of Danio rerio E7FBQ5/Naa80 towards actin-type N-termini.

(A) Overview of actin N-terminal processing leading up to Nt-acetylation. Class I actins are first co-translationally Nt-acetylated by NatB, and then actin methionine aminopeptidase removes the acetylated Met, before NAA80 acts on the acidic neo-N-terminus. Class II actins are processed by MetAP1/2 before Nt-acetylation by NatA and removal of an acetylated Cys by actin methionine aminopeptidase before Nt-acetylation by NAA80. In both humans and zebrafish, actin is conserved with most of the amino acid differences located to the N-terminus. (B) Acetylation assays with purified MBP- E7FBQ5/zNaa80 and different peptides derived from human proteins. (C) Acetylation assays with purified MBP- E7FBQ5/zNaa80 and different peptides derived from zebrafish actins.

The spatiotemporal expression of naa80 mRNA by whole-mount in situ hybridization and RT-qPCR.

(A, B, B′, C, C′, D, E, F) Whole-mount in situ hybridization was performed at different developmental stages of zebrafish embryos at 1 (A), 24 (B, B′), 48 (C, C′) and 72 (D) hpf. RT-qPCR at different embryonic stages (E) and adult tissues (F). For (A, B, B′, C′), dorsal view and anterior to the left. For (C, D), lateral view and anterior to the left. Fb, forebrain. Mb, midbrain. Hb, hindbrain. Pf, pectoral fin bud. Scale bar = 0.3 mm. For (E, F), each timepoint included biological triplicates as well as technical triplicates. (E, F) The expression levels were first normalized to the 18S housekeeping gene and the expression levels were compared with 18 hpf embryos (E) or compared with skin (F). Data shown are mean ± SD and mean values were shown on each bar.

Generation of stable mutant naa80 knockout lines. (A) Overview of naa80 mutant zebrafish crossing and experiments performed. (B) The Naa80 protein with location of 5del/1in and 13del mutations, as well as locations of the Ac-CoA binding motif (red) and proline-rich loop (yellow) highlighted. Also highlighted are the sense and missense regions and the premature stop codons which arise in the mutant alleles. (C) Representative PCR products (112 bp from WT allele, 99 bp from 13del allele) from naa80 13del fin clips visualized in agarose gel (4% agarose in lithium borate acetate buffer and GelRed).

Body length and weight are not affected by naa80 genotype.

Adult zebrafish with the indicated genotype were weighed and measured from rostrum to caudal tip. A one-way ANOVA test followed up with a Dunnett’s test was performed for the genotype comparison segregated by sex, where the +/+ values was selected as the control mean. *: P < 0.05, nonsignificant (ns): P > 0.05. Unpaired t test was performed for male versus female comparison. ****: P < 0.0001, ns: P > 0.05. The mean value for each dataset is indicated beneath the boxplot. (A) Body length values for males and females. (B) Measured weight of males and females with indicated sample size and mean. (C) The BMI for males and females was calculated by using the formula BMI=weight(kg)(body length (m))2.

Defective in vivo actin Nt-acetylation in naa80−/− zebrafish.

Mass spectrometry measurements of actin N-terminal peptides in cardiac muscle or skeletal muscle from the indicated genotypes. Log₂ intensity of the peptides is reported. Gray: not identified in the sample.

Zebrafish naa80 F0 KO larvae showed hearing loss-related phenotypes.

(A) Schematic representation depicts sensory tissues in zebrafish larva at 5–6 dpf including inner ear and neuromasts. Five sensory patches of inner ear including anterior crista (Ac), lateral crista (Lc), posterior crista (Pc), anterior macula (am), and posterior macula (pm). Schematic representation of lateral crista at lower right corner. Kinocilia and stereocilia of hair cell can be revealed by immunohistochemistry using anti-acetylated tubulin Ac-tub and phalloidin (F-actin), respectively. (B, C)naa80 F0 KO larvae have no obvious morphological abnormalities but showed smaller otoliths compared with Cas9-injected control larvae. (D) RT-qPCR showed a down-regulated naa80 expression in F0 KO (n = 3). Expression levels were normalized to 18S housekeeping gene and compared with the Cas9-injected controls. (E, F, E′, F′, E″, F″) Immunohistochemistry of both anti-acetylated tubulin (magenta, (E, F)) and phalloidin (cyan, E′, F′), and merged (E″, F″). (G) Quantification of hair cell numbers in the lateral crista of control (n = 3 larvae) and KO (n = 4 larvae) larvae. (H) Stereocilia length measurement in lateral crista hair cells of control and KO larvae. n = 31 stereocilia from three larvae for each group. (I) Evaluation of acoustic startle response. n = 48 larvae of each group. (J, K) Representative fluorescent images of Cas9-injected control (n = 12 larvae) and naa80 F0 KO (n = 13 larvae) larvae after Yo-Pro-1 uptake. Left panel (J, K) was whole-mount images after invert color into black and white, anterior to the left and dorsal to the top. Right panel (J′ and K′) was enlarged single neuromast. (L) Quantification of Yo-Pro-1 positive cells per neuromast. Two-tailed unpaired t test with Welch’s correction. **P < 0.01 and ****P < 0.0001.

Sequences of naa80 knockout alleles.

(A) The naa80 transcript, showing up- and downstream sequences (green), untranslated regions (red) and translated sequences (blue) of the introns, and parts of the 3,108 bp intron (gray). The location of the 13del mutation is shown by underlining, while the basepairs affected by the 5del/1in mutation is in yellow. The transcript sequence is also found in Table S1. (B) Alignment of parts of the naa80 CDS with the naa80 5del/1in and 13del alleles, showing the deletions and insertions (blue) and the locations of the new stop codons (red). The WT and mutant CDS and predicted protein sequences can be found in Table S1. (C) Representative DNA sequencing chromatograms for WT naa80 and hetero- and homozygous carriers (+/− or −/−) of either mutant.

Alignment of predicted protein sequences of Naa80, and the gene products of the naa80 5del/1in and naa80 13del alleles.