DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation - PubMed (original) (raw)
. 2016 Jul 7;99(1):115-24.
doi: 10.1016/j.ajhg.2016.05.002. Epub 2016 Jun 23.
Amanda J Walne 1, Mike Williams 2, Nicholas Bockett 1, Laura Collopy 1, Shirleny Cardoso 1, Alicia Ellison 1, Rob Wynn 3, Thierry Leblanc 4, Jude Fitzgibbon 5, David P Kelsell 6, David A van Heel 1, Elspeth Payne 7, Vincent Plagnol 8, Inderjeet Dokal 1, Tom Vulliamy 9
Affiliations
- PMID: 27346687
- PMCID: PMC5005432
- DOI: 10.1016/j.ajhg.2016.05.002
DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation
Hemanth Tummala et al. Am J Hum Genet. 2016.
Abstract
A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit.
Copyright © 2016. Published by Elsevier Inc.
Figures
Figure 1
Biallelic Mutations in DNAJC21 (A) Sanger sequencing traces and the genotype of each individual are given. Arrows indicate the mutated nucleotides. (B) The positions of the alterations caused by the mutations observed in our individuals are shown in the context of the conserved functional domains predicted in the DNAJC21 amino acid sequence. Residue numbers are given for the different domains. J domain refers to the DNAJ domain that defines this group of proteins; DBINO refers to the DNA-binding domain found on global transcription activator SNF2L1 proteins and chromatin re-modeling proteins. The frameshift p.Gly299Alafs∗2 variant is shown in red as it is predicted to arise from the deletion of exon 7 due to the splice-site mutation c.983+1G>T in individual 2. (C) Conservation of the HPD motif in the J domain of DNAJC21. Alignment of residues 16–50 of the human DNAJC21 J domain with four other DnaJ proteins was generated with ClustalW. The arrow indicates the proline residue that was mutated in individual 3. The aligned sequences are human (H.s.) DNAJC21 (UniProt:
Q5F1R6
), Drosophila melanogaster (D.m.) DNAJ-1 (UniProt:
Q53ZT0
), rice (O.s.) Oryza sativa DNAJ homolog (UniProt:
Q948S9
), Saccharomyces cerevisiae (S.c.) DnaJ-related protein SCJ1 (UniProt:
P25303
), and Escherichia coli (E.c.) chaperone protein DnaJ (UniProt:
P08622
). Asterisks indicate positions that have a single fully conserved residue, colons indicate conservation between groups of strongly similar properties, and periods indicate conservation between groups of weakly similar properties. (D) E. coli DnaJ domain topology (PDB:
1FAF
) visualized with Swiss PDB Viewer. Ribbon diagram depicts J domain (gray) and position of HPD motif (orange loop). The hydrogen bonds between residues in the HDP motif are indicated by dotted lines (black). Mimicking the individual 3 mutant by in silico analysis revealed loss of the proline’s cyclic ring, disrupting hydrogen bonds (dotted line, black), when substituted with alanine in the J domain.
Figure 2
Nucleolar Localization of DNAJC21 and Its Role in rRNA Biogenesis (A) DNAJC21 is present in both cytoplasmic (Cy) and nuclear fractions (Nu). Antibody against TBP was used as a control for nuclear fractions. WC, whole cell. (B) Immunocytochemistry shows the subcellular localization of DNAJC21 in different cell types. A lack of DNAJC21 immunostaining is observed in T cells from individual 4, whereas the parental T cells stain positive. Nucleophosmin (NPM1) is used as a control. Images display NPM1 (green), DNAJC21 (red), and DAPI (blue). Scale bar, 20 μm. (C and D) DNAJC21 translocates to the nucleus after actinomycin D treatment. NPM1 is used as positive control. (E) LCLs from individual 4 and asymptomatic heterozygous parent controls (1 and 2) were plated in the presence of increasing concentrations of actinomycin D for 48 hr and assayed for cell viability by staining with neutral red. Assays were performed in octuplets per experiment and repeated for a minimum of two independent experiments. Data points represent mean ± SEM. (F) Individual 4 LCLs show reduced expression for rRNA in nuclear extracts analyzed when compared to both those of parents and three unrelated samples as controls. Expression of SNORA63 and SNORA68 was determined as internal controls. All genes are normalized relative to expression of GAPDH mRNA. Data represent mean ± SD, n = 2, performed in triplicates.
Figure 3
DNAJC21 Associates with rRNA and Interacts with 60S Ribosome Maturation Factors (A) Expression of eGFP-tagged DNAJC21 wild-type and mutants was detected by immunoblotting with anti-GFP. GAPDH was used as a loading control. (B) Immunoprecipitation was performed with GFP-TRAP agarose beads and nuclear extracts of HeLa cells expressing wild-type and mutant forms of DNAJC21. Cells expressing EGFP alone were used as a control for the co-immunoprecipitation. RNA was extracted from the immunoprecipitates and analyzed by RT-PCR. Data represent mean ± SD, n = 2 independent experiments performed in duplicate. ∗p < 0.05, ∗∗∗p < 0.0001 Mann-Whitney U test. (C) Co-immunoprecipitations were performed in HeLa cells expressing eGFP or eGFP-DNAJC21. Lysates were treated with DNase I or RNase A prior to immunoprecipitation where indicated. Co-immunoprecipitated proteins were detected by western blot analysis with eGFP-, HSPA8-, ZNF622-, and PA2G4-specific antibodies. (D) Co-immunopreciptation from cells expressing eGFP-tagged wild-type and mutant forms of DNAJC21 reveal the variable interaction with cofactors involved in 60S ribosome maturation. IN, 10% input; IP, immunoprecipitate; WT, wild-type.
Figure 4
Loss of DNAJC21 Impairs Traffic of PA2G4, Corrupting Ribosome Biogenesis (A–C) Cytoplasmic accumulation of PA2G4 was observed in DNAJC21-knockdown cells and T lymphocytes from individual 4 when compared to relevant controls. (A) and (C) show DAPI (blue) and PA2G4 (red). Scale bars, 35 μm (A) and 20 μm (C). Panels indicate representative images taken from different fields of view in three separate experiments. (B) shows immunoblotting of cytoplasmic (C) and nuclear (N) fractions. (D) Immunoblotting of LCL lysates from individual 4 and parental controls for DNAJC21 and 60S maturation factors HSPA8, PA2G4, and ZNF622. (E) Cycloheximide-treated lysates from individual 4 and parent control LCLs, as well as DNAJC21 and non-target siRNA-treated HeLa cells, were fractionated on 15% to 45% sucrose gradients by ultracentrifugation. Absorbance at 254 nM across the gradient fraction is shown (top panels). Ribosomal subunit ratios (60S:80S) are indicated. TCA-precipitated proteins from equal aliquots of each fraction were immunoblotted for PA2G4 and RPL7 as a marker for 60S and 80S subunits and polysomes. Data presented is representative of two independent experiments (n = 2)
Figure 5
Loss of DNAJC21 Inhibits Cell Growth (A) After 72 hr of DNAJC21 siRNA by doxycycline (dox) treatment to induce shRNA targeting the 3′ UTR of DNAJC21 (100 ng/mL), whole-cell extracts were subjected to western blot analysis with DNAJC21- and GAPDH-specific antibodies. (B) FAC sorting plots show increased cell death in cells treated with DNAJC21 siRNA. (C) Phase contrast microscopy images of _DNAJC21_-RNAi-treated cells exhibiting abnormal morphology in comparison to controls. All panels are representative of images taken from different fields of view in two separate experiments. (D) T lymphocytes from individual 4 show impaired growth as compared to parental controls. Data represent mean ± SEM; ∗∗∗p < 0.001, Student’s t test. (E) Knockdown of endogenous DNAJC21 by dox administration is detected by western blotting using a mix of both DNAJC21 and GFP antibodies in stably transduced DNAJC21 3′ UTR shRNA HeLa cells. Arrows indicate positions of endogenous DNAJC21 and eGFP control. (F) Cell viability relative to an untreated dox (−) culture was measured by GFP fluorescence. This revealed the rescue of cell viability in the presence of eGFP-DNAJC21 upon dox induction of the shRNA targeting the endogenous transcript. Data represent mean ± SEM; ∗∗p < 0.01 one-way ANOVA with Tukey’s post hoc test. (G) In cells expressing eGFP alone, confocal imaging revealed cytoplasmic accumulation of PA2G4 and abnormal morphology upon dox administration. However, in cells expressing eGFP-DNAJC21, no defect in PA2G4 shuttling to the nucleus or cell morphology was observed upon dox addition (+) when compared to an untreated dox (−) culture. Images display eGFP (green) and PA2G4 (red). Panels are representative of images taken from different fields of view in three separate experiments. Scale bar, 20 μm.
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