moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans

Abstract

Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.

Figure 1. moc-6 is required for C. elegans Moco synthesis

(A) Amino acid alignment between human MOCS2A and C. elegans K10D2.7/MOC-6. Shaded amino acids (*) are identical. Strong (:) and weak (.) amino acid similarity are also displayed. (B) Nucleotide sequence for the wild type K10D2.7/moc-6 locus is displayed as are the sequences for newly generated K10D2.7/moc-6 deletions rae295, rae296, and rae297. Guide RNAs were designed targeting the 2 K10D2.7/moc-6 sequences displayed in red. The 2 corresponding PAM sites are displayed in blue. (C) Wild-type, rae295, rae296, and rae297 animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. (D) moc-6(rae296), cth-2(mg599); moc-6(rae296), and moc-6(rae296); cdo-1(mg622) mutant animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. For panels C and D, box plots display the median, upper, and lower quartiles, and whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. *, indicates a statistically significant difference (p<0.01, unpaired t-test) in C. elegans growth on ΔmoaA E. coli when compared to corresponding growth on wild-type E. coli. (E) Hatching rates for wild-type and mutant C. elegans was determined. Moco+ diet indicates that mothers were cultured on wild-type E. coli while Moco- indicates that mothers were shifted to ΔmoaA mutant E. coli at the L4 stage of development, depriving animals of dietary Moco during reproductive adulthood. n is the number of embryos scored for hatching for each genotype and condition. * (red), indicates a statistically significant difference (p<0.01, Chi-square test) in the hatching rates of the progeny of C. elegans mothers fed ΔmoaA E. coli when compared to the corresponding hatching rate when fed wild-type E. coli. * (black), indicates a statistically significant difference (p<0.01, Chi-square test) in hatching rates of the progeny of cth-2(mg599); moc-6(rae296) and moc-6(rae296); cdo-1(mg622) double mutant C. elegans mothers fed ΔmoaA E. coli when compared to moc-6(rae296) single mutant mothers fed ΔmoaA E. coli.