RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency

Abstract

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.

Fig. 1

RACK1 is essential for gastrulation. a Reconstruction of targeted RACK1 gene. The 142-bp deletion is at the level of the first loxP site (grey square) (scheme produced by MacVector software). b, c Resorbed knockout embryos at E9.5 (b, scale bar 1 mm) and their genotype identification (c). d Histological analysis of implantation sites showed the absence of the early egg cylinder and the presence of vacuolated areas (arrows) in one out of four E6.5 embryos (scale bar 80 μm)

Fig. 2

RACK1 is reduced in the presumptive ΔF/ΔF mouse embryo. Immunofluorescence analysis of E6.5 embryos, showing nuclear staining (Hoechst, blue) and RACK1 staining [13]. RACK1 immunohistochemical staining was virtually absent in 25 % of E6.5 embryos (bottom right). The residual fluorescence (arrow) is due to autofluorescence, visible also in the red channel (not shown and [64]). Scale bar 50 μm

Fig. 3

Belly spot phenotype in RACK1 mutant mice. a White belly spot and hypopigmented paws and tail tips in ΔF/+ adult mice. b Organ size is normal in ΔF/+ adults. c Representative RACK1 protein expression in brain, liver, spleen and kidney

Fig. 4

RACK1 mutant young females have delayed growth and reduced RACK1 protein levels. a, b Weight and head-to-anus length of 11-day-old pups (F females, M males; n = 17 per genotype; *p ≤ 0.05, **p ≤ 0.01). c Representative protein expression in brain, liver, spleen and kidney. d, e Quantitative PCR for RACK1 mRNA levels in brain and liver of littermates (n = 5 per genotype)

Fig. 5

snoRNA and rRNA production are not affected in mutant mice. a Northern blot analysis of snoRNAs in RNA samples from littermates, comparing wild-type and RACK1 ΔF/+ embryos (left ethidium bromide staining, right hybridization). The probes used are described in Materials and methods. b Representative Northern blot analysis for rRNA precursors (left ethidium bromide, right hybridization)

Fig. 6

RACK1 mutant MEFs have reduced PKC-stimulated translation. Global protein synthesis was measured by [³⁵S]methionine incorporation. Starved (st) MEFs were treated with (a) insulin (ins) and (b) PMA as described in Materials and methods (n ≥ 3 per genotype; *p ≤ 0.05, **p ≤ 0.01). c, d RACK1 mRNA and protein levels of the analyzed MEFs

Fig. 7

RACK1 mutant tissues show mild 80S accumulation. a, b Polysome profiles in 16-day-old females (+/+ versus ΔF/+ littermates). c Western blot analysis of mTORC1/2 pathway under the same conditions as in a and b

Fig. 8

p53 depletion slightly ameliorates the belly spot phenotype. a Induction of p53 stress by RACK1 depletion. 293 cells were transfected with siRNA in triplicate and p53 levels assayed by western blotting after 48 h. b Reduction in the pigmentation defect by p53 ablation. c Survival (Kaplan-Meier plot) of p53^−/− mice in RACK1 wild-type versus mutant background (n = 16 p53^−/− RACK1^+/+ males; n = 23 p53^−/− RACK1^ΔF/+ males)