Fig 2: Generation of Hcfc1 and Ronin point mutants.a Chromatograph traces of wild type and Hcfc1 A115V hemizygous male mice showing the c.344 C>T (p.Ala115Val) mutation and a silent mutation (c.351 T>A) creating a BccI restriction enzyme site. b Amino acid structural change due to A115V. c Image of the predicted A115V substitution within the HCFC1 protein. d Alanine 115 in HCFC1 is evolutionarily conserved through vertebrates. e Mouse HCFC1 structure showing the localization of the A115V mutation. f Ratios of Hcfc1A115V/Y male progeny at E16.5, P0, and weaning. Statistically significant differences in the observed genotypes were determined using the chi-squared test (?2 = 53.13, ***p = 3.12 × 10-13). g Chromatograph traces of wild type and Ronin F80L homozygous mice showing the c.240 C>G (p.Phe80Leu) mutation. h Amino acid structural change due to F80L. i Image of the predicted F80L substitution within the RONIN protein. j Phenylalanine 80 in RONIN is evolutionarily conserved through vertebrates. k Mouse RONIN structure showing the localization of the F80L mutation. l Ratios of RoninF80L/F80L progeny at E18.5 (C-sectioned) and weaning. Statistically significant differences in the observed genotypes at weaning were determined using the chi-squared test (?2 = 89.68, ***p = 2.79 × 10-21).

Fig 3: Cobalamin-related metabolic perturbations in Hcfc1A115V/Y and RoninF80L/F80L mice.a RONIN binding motif within the Mmachc promoter. b RONIN ChIP-PCR of the Mmachc promoter. c QrtPCR analysis (n = 3 per genotype) of Mmachc in the Hcfc1A115V/Y and RoninF80L/F80L embryonic brains. d Western blot analysis of MMACHC in the Hcfc1A115V/Y and RoninF80L/F80L embryonic brains. e [57Co] cyanocobalamin incorporation assay (n = 8 wild type and n = 5 mutants) to determine cobalamin distribution in MEFs. f 5-[14C] methyltetrahydrofolate (methylTHF) incorporation assay of methionine synthase (MTR) activity in MEFs (n = 3 or 4 wild type and n = 3 mutants). g [14C] propionate incorporation assay of methylmalonyl-CoA mutase activity in MEFs (n = 3 or 7 wild type and n = 3 or 7 mutants). h Plasma levels of methylmalonic acid (MMA), homocysteine (Hcy), and methionine (n = 5 or 7 wild type and n = 5 or 7 mutants). i Summary of MMACHC-dependent metabolic deficiency identified in Ronin and Hcfc1mutant mice. RONIN F80L and HCFC1 A115V mutant protein function is compromised leading to reduced Mmachc transcription and protein activity (1 and 2). This defect results in reduced production of MeCbl and reduced MTR activity (3), and the corresponding buildup of the upstream metabolite homocysteine (4). Reduced MMACHC also leads to lower levels of AdoCbl production and MUT activity (5 and 6), and the buildup of methylmalonyl-coA (7). All data are shown as mean ± SEM and n = 3 biologically independent samples per genotype. Statistically significant differences between genotypes were determined using the t test (two-tailed). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source data file.

Fig 4: Ribosomopathy phenotypes in Hcfc1 and Ronin mutants.a–d Crosses and penetrance of ribosomopathy-associated phenotypes. e White belly spotting phenotype. f Homeotic transformation of C7 to T1 indicated by an ectopic rib on C7 (arrows). g, h Kinked tails in Ronin+/F80L; Hcfc1A115V/Y embryos and a single Ronin+/F80L mouse. i Hypopigmented paws on Hcfc1A115V/Y adults. j Exencephaly in RoninF80L/F80L embryos. k Polydactyly in a Ronin+/F80L; Hcfc1+/A115V mouse. l Summary of phenotypes reported in single ribosome protein mutants. Phenotypes listed in red were observed in Ronin and Hcfc1 mutants, but never in wild type mice. Panel l was created with BioRender.com.