Biallelic mutations in WRAP53 result in dysfunctional telomeres, Cajal bodies and DNA repair, thereby causing Hoyeraal-Hreidarsson syndrome

Abstract

Approximately half of all cases of Hoyeraal-Hreidarsson syndrome (HHS), a multisystem disorder characterized by bone marrow failure, developmental defects and very short telomeres, are caused by germline mutations in genes related to telomere biology. However, the varying symptoms and severity of the disease indicate that additional mechanisms are involved. Here, a 3-year-old boy with HHS was found to carry biallelic germline mutations in WRAP53 (WD40 encoding RNA antisense to p53), that altered two highly conserved amino acids (L283F and R398W) in the WD40 scaffold domain of the protein encoded. WRAP53β (also known as TCAB1 or WDR79) is involved in intracellular trafficking of telomerase, Cajal body functions and DNA repair. We found that both mutations cause destabilization, mislocalization and faulty interactions of WRAP53β, defects linked to misfolding by the TRiC chaperonin complex. Consequently, WRAP53β HHS mutants cannot elongate telomeres, maintain Cajal bodies or repair DNA double-strand breaks. These findings provide a molecular explanation for the pathogenesis underlying WRAP53β-associated HHS and highlight the potential contribution of DNA damage and/or defects in Cajal bodies to the early onset and/or severity of this disease.

Fig. 1. Identification of missense mutations in WRAP53 in a patient with HHS.

a At an age of 2.7 years, the proband demonstrated microcephaly, short stature, broad gait, fine blond hair and dysmorphic features (including strabismus, epicanthal folds, cup-shaped protruding overfolded ears, a depressed nasal tip and widely spaced teeth). b Analysis of telomere lengths by quantitative PCR in peripheral blood leukocytes collected from the proband at this same age (solid square). The reference relative telomere length (RTL) were determined from telomere length analysis of blood leukocytes from 173 healthy subjects (0–84 years of age; open circles). The curves shown depict the first, 10th, 50th, 90th, and 99th normal percentiles at each age. c Schematic illustration of the WRAP53 gene, the protein encoded and the positions of the mutations detected in the proband. The DC mutations in WRAP53β reported previously are also marked with the superscripts indicating mutations that occur in the same patient. Note: Exon numbering is based on the GenBank sequence DQ431240, i.e., the separation of exon 1β and 2 by an intron. In the reference sequence NM 018081 this intron is included resulting in different exon numbering. d Conservation of the sites of HHS mutations (marked in red) among species. e Pedigree of the family carrying autosomal recessive HHS and mutations in WRAP53. The arrow indicates the proband. The heterozygous carrier (the father, half-filled symbol), compound heterozygous carrier (the proband, filled symbol), and wild-type individual (the mother, non-filled symbol) are shown.

Fig. 2. WRAP53β mutants associated with HHS are expressed at reduced levels and with an altered localization, disrupting the structure of Cajal bodies.

a HeLa cells were transfected with a vector encoding: EGFP only (GFP-Empty), or EGFP-tagged wild-type (WT) or mutant (L283F or R398W) WRAP53β for 24 h and thereafter subjected to western blot for GFP and GAPDH. b Quantitative PCR analysis of the levels of mRNA encoding EGFP alone (GFP-Empty), EGFP-tagged wild-type (WT) or mutant (L283F or R398W) WRAP53β in HeLa cells 24 h post-transfection. Primers targeting the GFP sequence were utilized to avoid detection of endogenous WRAP53β. The values shown represent the RNA levels normalized to β-actin and relative to the levels of GFP-WT WRAP53β. c HeLa cells were transfected with a vector encoding: EGFP-tagged wild-type (WT) or mutant (L283F or R398W) WRAP53β for 16 h followed by addition of the protease inhibitor MG132 (10 μM) for another 8 h, as indicated. GFP, p53, and GAPDH were detected by western blot. The levels of p53, a protein with rapid turnover via the proteasome, was used as a control for proteasome inhibition by MG132. The numbers in black represent densitometric quantification of each protein normalized to GAPDH and relative to the levels of wild-type WRAP53β (for the GFP proteins) or p53 (for the p53 protein) in cells not treated with MG132 (first lane). d The same densitometric numbers as shown in c, including error bars. Again, the values represent the GFP levels relative to the levels of wild-type WRAP53β. e Immunofluorescent detection of the GFP-tagged WRAP53β proteins indicated in HeLa cells 24 h post-transfection. The Cajal bodies were visualized by immunostaining for coilin and nuclei stained blue with DAPI in all cases. f Quantification of cells as shown in e exhibiting nuclear enrichment of the GFP-tagged wild-type or mutant WRAP53β. The bars indicate the percentage of 100 GFP-positive cells in each experiment in which the nuclear signal was at least as intense as the signal in the cytoplasm (i.e., with a nuclear/cytoplasmic signal intensity ratio ≥1). g Quantification of cells as shown in e in which GFP accumulated in Cajal bodies. The bars indicate the percentage of 100 cells in each experiment that stained for both GFP and Cajal bodies and in which these bodies were enriched for GFP. h Quantification of cells as shown in e containing Cajal bodies. The bars indicate the percentage of 100 GFP-positive cells whose nuclei also contained Cajal bodies (i.e., formation of coilin foci). In all cases, the values are means ± s.d. (the error bars) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns (not significant) as determined by Student’s t-test.

Fig. 3. HHS mutants of WRAP53β interact less with components of Cajal bodies and telomerase but more with those of the TRiC complex.

a HeLa cells were transfected with the GFP proteins indicated for 24 h and then subjected to immunoprecipitation with a GFP antibody, followed by immunoblotting. b Densitometric quantifications of western blots as shown in a. The bars represent the levels of co-precipitated protein normalized to the levels of the corresponding immunoprecipitated GFP protein itself and then relative to the value obtained in wild-type WRAP53β (i.e., levels of (co-precipitated protein/immunoprecipitated protein itself)/(co-precipitated protein by wild-type WRAP53β/immunoprecipitated wild-type WRAP53β itself)). The values are means ± s.d. (the error bars) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns (not significant) as determined by Student’s t-test. c HeLa cells were treated as in a, followed by purification of RNA and assessment of the levels of co-precipitated RNA employing qPCR. The values show the amount of co-precipitated RNA as a percentage of input with the error bars depicting the s.d. (n = 3 for scaRNA5 or 4 for TERC, scaRNA2 and actin). *p < 0.05, ***p < 0.001, ns (not significant) as determined by Student’s t-test.

Fig. 4. HHS-associated WRAP53β mutations attenuate repair of DNA double-strand breaks and lead to accumulation of DNA damage.

a HeLa cells were transfected with the GFP plasmids indicated and Flag-RNF8 for 24 h; irradiated with  2 Gy; left to recover for 1 h; and then subjected to immunoprecipitation with a GFP antibody, followed by immunoblotting. b Densitometric quantifications of western blots as shown in a. The bars represent the levels of co-precipitated protein normalized to levels of the corresponding immunoprecipitated GFP protein itself, and then relative to the value obtained in wild-type WRAP53β. The values are means ± s.d. (the error bars) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns (not significant) as determined by Student’s t-test. c, d HeLa cells were transfected with siControl or siWRAP53#2 oligonucleotides for 24 h; followed by transfection with GFP-Empty or GFP-WRAP53β^{siRNA resistant} plasmids for another 24 h; irradiated with 2 Gy; and 1 h later harvested for either western blotting using the antibodies indicated (c) or immunostaining for 53BP1 (d). The graph in d shows the percentage of 100 GFP-transfected cells in each experiment whose nuclei contained ≥10 53BP1 foci with the error bars depicting the s.d. (n = 4). e HeLa cells were transfected with siRNA for 8 h; followed by transfection with the GFP plasmids indicated for another 16 h; irradiated with 2 Gy; and after 24 h of recovery, immunostained for γH2AX. The graph shows the percentage of 100 GFP-transfected cells in each experiment whose nuclei contained ≥10 γH2AX foci with the error bars depicting the s.d. (n = 3). *p < 0.05, **p < 0.01, ns (not significant) as determined by Student’s t-test.

Fig. 5. Model of defects caused by HHS mutations in WRAP53.

Schematic illustrations of the normal folding and functions of wild-type (WT) WRAP53β and molecular defects and functional consequences of the HHS mutations. WRAP53 mutations results in misfolding and destabilization of the WRAP53β protein and impair the binding to and trafficking of partner proteins and RNAs to correct cellular sites. Consequently, this perturbs maintenance of Cajal bodies, shortens telomeres and impairs DNA repair, thereby causing HHS.