Figure 1.
Characterization and genetic identification of the rep mutation.
A 15days post-partum rep homozygous mice (right) displayed reduced pinna compared to control littermates (left). B Haplotype analysis of 81 mutant mice derived from the outcross-intercross strategy. Markers are shown with their position (cM) on chromosome 2 (Ensembl V50). The C57BL/6J alleles are shown with black boxes whereas the white boxes indicate the presence of the C3HeB/FeJ allele. Haplotypes with the same allelic distribution were collected and their number is given at the top of each column. C Sequence chromatograph spanning the rep mutation site compared with that of a wild-type control.
Figure 2.
Growth curves of the rep homozygote mutants and control littermates.
Body weight curves of rep/rep individuals generated from heterozygote intercrosses (male rep/rep n = 9; male wt n = 15; female rep/rep n = 12; female wt n = 22) reveal that growth of mutant homozygotes is affected during post-natal development compared to control littermates.
Figure 3.
Analysis of the gonads of rep/rep mutant mice.
A, B Histological analysis of hematoxylin-stained sections. In ovaries, all stages of folliculogenesis from primordial to preovulatory follicles and corpus luteum were observed in mutant mice (A) as in wild-type (B), suggesting that ovaries are functional. Scale bar = 150 µm. C Histology of the testis from adult rep mutant male. All stages of spermatogenesis were visible, and no alteration of tubule diameter was observed. Scale bar = 50 µm. D Histology of the cauda epididymis from adult rep mutant male. No abnormalities were observed. Scale bar = 20 µm.
Figure 4.
Morphological variations observed in skulls and mandibles of wt, rep and Tm1Gsc mice.
A Major cranial anomalies observed in Prkrarep and Prkratm1Gsc mice. Arrows indicate the principal defects for each mutant, i.e. the reduced coronoid process and mandibular condyle, and the short nasal bone in rep mice and the bregmatic fontanelle-like structure of Prkratm1Gsc mice. Scale bar: 5 mm. B Plot of principal components 1 and 2 based on Procrustes analysis of 3D landmark coordinates of wt, rep and Prkratm1Gsc mice. Squares: wt mice; Triangles: Prkratm1Gsc mice; Filled diamonds: rep heterozygous mice; Open diamonds: rep homozygous mice. On the principal components analysis (PCA) performed from cranial landmarks (top panel), PC1 represents 54.7% of variance and PC2 15.1%. On the PCA performed from mandibular landmarks (bottom panel), PC1 represents 46.0% of variance and PC2 16.1%.
Table 1.
Weights of adult rep mice compared to those of WT controlsa.
Figure 5.
Analysis of RAX mRNA expression in the brains of rep mice.
Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure 6.
Functional characterization of the RAX (S130P) mutant.
A DsRNA electrophoretic mobility shift assay: Purified His-RAX or His-RAX (S130P) was incubated at the indicated concentration with 5′end labelled dsRNA. In the indicated lanes, poly(I:C) was added to the reaction as a competitor to demonstrate dsRNA-binding specificity. B DsRNA-pull-down assay: Purified His-RAX or His-RAX (S130P) was incubated with 5′end labelled dsRNA. His-RAX was pulled-down from the reactions with Ni-NTA agarose and bound dsRNA was measured by liquid scintillation (expressed in counts per minute). CPM bound to BSA control has been subtracted from all lanes as background. C Dimerization assay: Purified His-RAX or His-RAX (S130P) was incubated with lysates from L929 cells expressing empty provirus, FLAG-RAX or FLAG-RAX (S130P). His-RAX was pulled-down from the reactions with Ni-NTA agarose and incubated with micrococcal nuclease to eliminate dsRNA-facilitated protein-protein interactions. Samples were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies. Total input lysates were directly analyzed for measuring the levels of expression of the indicated proteins. D PKR interaction assay: FLAG-RAX was immunoprecipitated with an agarose-conjugated antibody against FLAG from lysates prepared from unstressed or arsenite treated L929 cells expressing empty provirus, FLAG-RAX or FLAG-RAX (S130P). Immunoprecipitated samples were incubated with micrococcal nuclease to eliminate dsRNA-facilitated protein-protein interactions. Samples were resolved by SDS-PAGE and PKR was detected by western blot to measure RAX-PKR interaction (top panel: PKR is the upper band and IgG is the lower band (denoted by *)). Other panels show the levels of FLAG-RAX in the immunoprecipitates and the levels of PKR and actin in the input lysates. E PKR activation assay: eIF2α phosphorylation was monitored by western blotting with a phospho-eIF2α-specific antibody (top panel). The middle and the bottom panels show the levels of eIF2α and RAX respectively. In these experiments, expression of endogenous RAX had been ablated by expressing a shRNA directed toward the 3′UTR of the mRNA. In those cells, WT or mutant RAX was ectopically expressed using lentivirus vectors; cells were stressed by treating them with sodium arsenite (100 µM), where indicated.
Figure 7.
Reduced ectopic expression of RAX (S130P) in L929 cells.
A RAX mRNA levels: Realtime RT-PCR analyses were used to determine the levels of FLAG-RAX mRNA relative to 18S rRNA using RNA samples isolated from the indicated cells. B RAX protein levels: FLAG western blot of L929 cells infected with empty lentivirus, or lentivirus encoding a provirus to ectopically express FLAG-RAX or FLAG-RAX (S130P) at the indicated MOI. Three times more virus was required for FLAG-RAX (S130P) to achieve protein levels comparable to WT. C RAX turnover analyses: Cells were treated with cycloheximide to inhibit de novo protein synthesis, cells lysates were prepared at the indicated time points and protein levels were measured by Odyssey quantitative western blot of FLAG- RAX and FLAG-RAX (S130P) using actin as the internal control. D Normalized levels of RAX: FLAG-RAX signal was normalized to that of actin and plotted at the indicated times following cycloheximide treatment.
Figure 8.
Lower levels of RAX protein in the brains of rep mice.
A RAX levels in proteins recovered from the same samples used for RAX mRNA measurements: Protein re-precipitated from trizol extracts of the same brain preparations shown in Figure 5 was analyzed by western blot for RAX and actin expression. B RAX levels in conventional protein extracts of brains: RAX western blot from brains homogenized and lysed in detergent-lysis buffer (see materials and methods). RAX expression was analyzed in wild-type, Prkratm1Gsc (denoted −/−) and rep (denoted 3) mice.