Fig 2: Zebrafish eif2b5 mutant alleles show intron retention, activated ISR, and role of truncated Eif2b5 in worsening phenotype.(A) Schematic showing integrated stress response activated intron 12 retention of eif2b5 resulting in premature stop codon and truncated form of EIF2B5. (B) Fold change of eif2b5 intron 12 expression with qRT-PCR in eif2b5zc103/zc103, eif2b5zc102/zc102, and eif2b2sa17223/sa17223 mutants relative to controls. (C) qRT-PCR for intron 12 expression in a VWM patient and their control unaffected father. (D) Control-injected larvae, compared to those injected with truncated eif2b5 construct, shows impaired swimming behavior; distance moved, time spent moving, and velocity, at 5 dpf. (E) qRT-PCR for atf4, bip, chopII, and perk ISR transcripts shows increased expression in eif2b5zc103/zc103, eif2b5zc102/zc102, and eif2b2sa17223/sa17223 mutants. (F) qRT-PCR for ISR transcripts (atf 4, bip, chop II, and perk) shows increased expression following injection of truncated eif2b5 construct. Figure 8—source data 1.qRT-PCR for intron 12 expression changes (eif2b5, zebrafish). Figure 8—source data 2.qRT-PCR for intron 12 expression changes (eif2b2, zebrafish). Figure 8—source data 3.qRT-PCR for intron 12 expression changes (human). Figure 8—source data 4.Behavior data for truncated eif2b5 effects. Figure 8—source data 5.qRT-PCR for ISR transcript expression. Figure 8—source data 6.qRT-PCR for ISR transcript expression changes following injection with truncated eif2b5.

Fig 3: Feed-forward effect of truncated EIF2B5.(A–C) Under normal conditions, stress leads to activation of the ISR, which is terminated by dephosphorylation of eIF2a by PP1 (protein phosphatase one complex) and resumption of normal cap-dependent translation. (A’–C’) In the presence of eif2b subunit mutations in VWM, normal translation is impaired, and there is activation of the ISR above baseline (A’). In response to stress, there is ISR activation and expression of truncated EIF2B5, which leads to chronic activation of the ISR and an inability to terminate the ISR.

Fig 4: Zebrafish eif2b allele generation and characterization.(A-D) Depiction of zebrafish eif2b subunits exon structure and the location and nucleotide change for each mutant. (A) eif2b1 harbors a T/A transversion resulting in an early stop in exon 8. (B) eif2b2 has a G/A transition in exon 5, mutating an essential splice site. (C) eif2b4 has a G/A transition in exon 12 mutating an essential splice site. (D) eif2b5 exon one was targeted for mutagenesis using a gRNA (red). Six distinct alleles were recovered (described in text). (E) Chromatograms of cDNA confirm presence of predicted mutations for eif2b5zc103/zc103, eif2b5zc102/zc102, and eif2b2sa17223/sa17223. (F) eif2b5zc103/zc103 mutants survive until adulthood in Mendelian ratios, but show grow defects compared to their heterozygous and wild-type siblings. (G) Adult eif2b5zc103/zc103 lengths are significantly shorter compared to their wild-type and heterozygous siblings. (H) Bright-field (BF) images of 6 dpf eif2b5zc103/zc103, eif2b5zc102/zc102, and eif2b2sa17223/sa17223 larva. eif2b2sa17223/sa17223 and eif2b5zc102/zc102 have no swim bladder (arrowhead) and a small head. (I) Kaplan-Meyer survival curves from an eif2b2sa17223/+ heterozygous in-cross shows 1% (n = 3) homozygote survival at 10 dpf (total n = 302); however no homozygotes live past 2 weeks of age. (J) Kaplan-Meyer survival curves from an eif2b5zc102/+ heterozygous in-cross shows that all homozygotes were dead by 10 dpf (total n = 62). (K) Kaplan-Meyer survival curves from an eif2b5zc103/+ heterozygous in-cross show no mortality of homozygotes. (L) Motor swimming analysis shows impaired swimming behavior in mutants. Distance moved, time spent moving, and velocity, for wild-type controls, and eif2b5zc103/zc103, eif2b5zc102/zc102, and eif2b2sa17223/sa17223 mutants, at 5, 6, and 7 dpf. Mean shown with 95% confidence intervals. Figure 2—source data 1.Quantification of lengths. Figure 2—source data 2.Quantification of behavior results. Figure 2—source data 3.Quantification of behavior results of eif2b4sa17367/sa17367 allele. Figure 2—source data 4.qRT-PCR for ISR transcripts for eif2b4sa17367/sa17367 allele. Figure 2—source data 5.Survival quantification for eif2b4sa17367/sa17367 allele. Figure 2—source data 6.Survival quantification for eif2b5zc103/zc103allele.