Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita

Abstract

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

Figure 5. Telomere lengths of cases harboring PARN mutations.

(A) Telomere lengths in family 1 were measured by automated multicolor flow-FISH and depicted as percentiles by calculating a reference range for telomere length over age in lymphocytes from 400 healthy individuals. Both of the affected children (indicated by red circles) show markedly shortened telomere lengths below first percentile for age in both lymphocyte and granulocyte lineages, while the parents (depicted by black triangles) have telomere lengths in the normal range. (B) Relative telomere lengths of cases 1–3 are reduced compared with controls. Cases 1–3 were analyzed by the MMqPCR method and compared with controls (n = 46). The dotted line represents the first percentile. Blue circles represent controls. Red squares represent cases. **P < 0.01, Mann-Whitney U test.

Figure 4. PARN deficiency effects telomere biology.

(A) Cases of PARN deficiency show reduced levels of expression of 4 telomere biology genes in blood. The relative expression of each gene, after normalizing to β-actin, is shown. Samples from cases 1-3 were analyzed, (one sample each from cases 1 and 2, and two independent samples from case 3; n = 3 cases) and compared with controls (n = 19). Bars represent the median relative expression ratio (*P < 0.05, **P < 0.01 Mann-Whitney U test). (B) Case 1 LCLs show reduced levels of expression of 4 telomere biology genes. All genes are normalized to β-actin and are expressed relative to the father’s sample. Data represent mean ± SD, n = 3. (C) Following 0, 1, 2, and 4 hours actinomycin D treatment, mRNA half-life (t_1/2) calculated as indicated in hours for each gene is determined in GFP and PARN siRNA knockdown samples. Data represent mean ± SEM, n = 2. (D) Reduced dyskerin and TRF1 protein levels in case 1 LCLs and PARN siRNA–treated cells; β-actin is used as a loading control.

Figure 3. Lack of nuclear deadenylation and abnormal DNA damage response in PARN-deficient cells.

(A and B) LCLs were exposed to UV light (40 J/m²) and allowed to recover over the time points indicated. Nuclear extracts from these cells were then tested for deadenylase activity using a fluorescent A9 substrate in a gel-based assay. The upper arrow (A9) indicates intact RNA substrates containing nine 3′ adenosine residues, the lower arrow (A1) indicates the reaction product containing a single 3′ adenosine residue, and the * denotes related deadenylated products. (B) Fluorescence-based assay shows reduced deadenylation kinetics in case 1 LCL nuclear extracts upon UV stress over time when compared with father and control. (C) Immunoblotting using an anti-p53 antibody in case 1 LCLs compared with his father and an unrelated control. Lamin A/C is used as loading control for nuclear lysates. (D) Densitometric analysis of the data in C shows relative changes in p53 expression at the indicated time points relative to the 0-hour time point in case 1 LCLs compared with both the father and an unrelated control after exposure to UV light. (E) Compared with his father and an unrelated control, case 1 LCLs showed reduced survival 48 hours after UV treatment (n = 3). (F) Cell-cycle abnormalities in case 1 LCLs compared with his father and an unrelated control showed there is a significant increase in the proportion of viable cells in G₂/M 48 hours after treatment (n = 3). In all cases, data represent mean ± SEM, **P < 0.01; ***P < 0.0001 1-way ANOVA with Tukey’s post hoc test.

Figure 2. Mutations in PARN cause deadenylation deficiency.

(A and B) Immunocytochemistry and Western blotting on EBV-transformed LCLs revealed no apparent differences in localization or expression of PARN between control, father, and case 1. Antibody against TATA binding protein (TBP) was used as a loading control. Immunostaining images show DAPI (blue) or PARN (red) from case 1, father, or control. Scale bar: 8 μm. (C) A gel-based deadenylase assay shows reduced deadenylation activity of an RNA substrate in whole-cell extracts from case 1 LCLs and PARN siRNA–treated (PARNsi-treated) HEK293 cells. The upper arrow (A9) indicates intact RNA substrates containing nine 3′ adenosine residues, the lower arrow (A1) indicates the reaction product containing a single 3′ adenosine residue, and * denotes related deadenylated products. (D and E) Fluorescence-based measurement shows reduced deadenylation kinetics in case 1 LCLs and PARNsi-treated cells. (F–H) PARN topology shows the position of p.Ala383Val missense change in the ND2 domain. (F) In silico analysis of PARN catalytic site (PDB 2A1R) denotes the amino acids from each promoter (ball and stick model; green) and bound RNA poly(A) (black). The mutated alanine 383 (Ala 383) residue is shown in blue. The adjacent aspartic acid 382 (Asp 382) residue has been shown to be involved in RNA poly(A) processivity (19). (G) Ribbon diagram (yellow) showing part of PARN nuclease domain and RNA poly(A) complex (black). (H) The missense change Ala 383 (blue, in G) to valine 383 (Val 383) (red) introduces a side chain (arrow head) in the α-helices (yellow) of PARN nuclease domain.

Figure 1. Identification of biallelic mutations in PARN.

(A–C) Sanger sequencing traces confirm the presence of the mutations identified by exome sequencing in cases 1-4. Pedigrees are also shown. A representative trace of the variant is shown for parents and cases. The gray shading of the parents in DCR373 indicates that they are predicted to be heterozygous. (D–G) Photographs of case 3 showing some of the clinical features: (D) abnormal dentition and abnormal facial features, including dysmorphic ears and microcephaly; (E) sparse hair, (F and G) nail dystrophy. (H) A linear diagram of the PARN protein shows functional domains and the effect of mutations identified in cases 1-4 giving rise to relevant protein variants. ND1 and ND2, nuclease domain 1 and nuclease domain 2; R3H, conserved arginine and 3-histidine containing domain; RRM, RNA recognition motif; NLS, nuclear localization signal; CTD, C-terminal domain.