Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita
Hemanth Tummala, Amanda Walne, Laura Collopy, Shirleny Cardoso, Josu de la Fuente, Sarah Lawson, James Powell, Nicola Cooper, Alison Foster, Shehla Mohammed, Vincent Plagnol, Thomas Vulliamy, Inderjeet Dokal
Hemanth Tummala, Amanda Walne, Laura Collopy, Shirleny Cardoso, Josu de la Fuente, Sarah Lawson, James Powell, Nicola Cooper, Alison Foster, Shehla Mohammed, Vincent Plagnol, Thomas Vulliamy, Inderjeet Dokal
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Research Article Aging Genetics Hematology

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.

Authors

Hemanth Tummala, Amanda Walne, Laura Collopy, Shirleny Cardoso, Josu de la Fuente, Sarah Lawson, James Powell, Nicola Cooper, Alison Foster, Shehla Mohammed, Vincent Plagnol, Thomas Vulliamy, Inderjeet Dokal

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Figure 3

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

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Lack of nuclear deadenylation and abnormal DNA damage response in PARN-d...
(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.