Difference in an intermolecular disulfide-bond between two highly homologous serum proteins Leg1a and Leg1b implicates their functional differentiation.

Zebrafish Liver-enriched gene 1a (Leg1a) and Leg1b are liver-produced serum proteins encoded by two adjacently linked homologous genes leg1a and leg1b, respectively. We previously showed that maternal-zygotic (MZ) leg1a null mutant developed a small liver at 3.5 days post-fertilization (dpf) during winter-time or under UV-treatment and displayed an abnormal stature at its adulthood. It is puzzling why Leg1b, which shares 89.3% identity with Leg1a and co-expressed with Leg1a, cannot fully compensate for the loss-of-function of Leg1a in the leg1azju1 MZ mutant. Here we report that Leg1a and Leg1b share eight cysteine residues but differ in amino acid residue 358, which is a serine in Leg1a but cysteine (C358) in Leg1b. We find that Leg1b forms an intermolecular disulfide bond through C358. Mutating C358 to Methionine (M358) does not affect Leg1b secretion whereas mutating other conserved cysteine residues do. We propose that the intermolecular disulfide bond in Leg1b might establish a rigid structure that makes it functionally different from Leg1a under certain oxidative conditions.

Upf3a but not Upf1 mediates the genetic compensation response induced by leg1 deleterious mutations in an H3K4me3-independent manner.

Genetic compensation responses (GCRs) can be induced by deleterious mutations in living organisms in order to maintain genetic robustness. One type of GCRs, homology-dependent GCR (HDGCR), involves transcriptional activation of one or more homologous genes related to the mutated gene. In zebrafish, ~80% of the genetic mutants produced by gene editing technology failed to show obvious phenotypes. The HDGCR has been proposed to be one of the main reasons for this phenomenon. It is triggered by mutant mRNA bearing a premature termination codon and has been suggested to depend on components of both the nonsense mRNA-mediated degradation (NMD) pathway and the complex of proteins associated with Set1 (COMPASS). However, exactly which specific NMD factor is required for HDGCR remains disputed. Here, zebrafish leg1 deleterious mutants are adopted as a model to distinguish the role of the NMD factors Upf1 and Upf3a in HDGCR. Four single mutant lines and three double mutant lines were produced. The RNA-seq data from 71 samples and the ULI-NChIP-seq data from 8 samples were then analyzed to study the HDGCR in leg1 mutants. Our results provide strong evidence that Upf3a, but not Upf1, is essential for the HDGCR induced by nonsense mutations in leg1 genes where H3K4me3 enrichment appears not to be a prerequisite. We also show that Upf3a is responsible for correcting the expression of hundreds of genes that would otherwise be dysregulated in the leg1 deleterious mutant.