Posttranscriptional modulation of TERC by PAPD5 inhibition rescues hematopoietic development in dyskeratosis congenita

Abstract

Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERC levels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERC could improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3 or PAPD5 levels in DKC1 mutant hESCs led to functional improvements in TERC levels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5 inhibition is sustained in differentiated CD34+ cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5 is a potential strategy to reverse hematologic dysfunction in DC patients.

Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita

“…These protocols recapitulate, in vitro, the major aspects of blood development in vivo, 24 a strategy that we and others have shown to accurately model key aspects of DC. 18,21,25,26 A schematic of our protocol is depicted in supplemental Figure 4. Our data show that although CD34 1 CD43early hematopoietic progenitors (day 8 of differentiation) ( Figure 2E-F) were similar in all samples, definitive hematopoietic colony potential analysis (day 28 of differentiation) revealed that treatment with different concentrations of RG7834 significantly increased the hematopoietic potential of DKC1_A353V cells ( Figure 2G).…”

“…13,14 We and others have shown that the degradation of TERC by the exosome can be inhibited by reducing the 39-end oligoadenylation of TERC through the modulation of PAPD5 [noncanonical poly(A) polymerase 5] levels. [13][14][15][16][17] Utilizing the targeted differentiation of human embryonic stem cells (hESCs), we showed that the genetic silencing of PAPD5 is able to improve the hematopoietic potential of DC cells, 18 thereby rescuing the major phenotype observed in this disease. This finding opens the possibility that the chemical inhibition of PAPD5 by a safe, efficient, orally available compound could represent a novel alternative to be pursued in the clinical management of patients with DC and mutations that impair TERC biology.…”

Regulation of human telomerase in homeostasis and disease

“…PABPN1-unbound oligo-adenylated hTR intermediates are subject to degradation by the recruitment of the nuclear RNA exosome by the nuclear exosome targeting (NEXT) complex in conjunction with the cap binding complex (CBC) 63,71 . Depletion of exosome components, NEXT or CBC can rescue the phenotype of low hTR levels owing to dyskerin loss or hTR point mutation-sin the H/ACA domain, suggesting that the exosome serves also to remove defective or misassembled hTR transcripts [71][72][73] . Early during transcription, telomerase RNAs acquire the m 7 G monomethylguanosine cap, which is hypermethylated to a m 2,2,7 G trimethylguanosine cap by the enzyme trimethylguanosine synthase (TGS1) 74,75 (FIG.…”

Telomere Dysfunction Activates p53 and Represses HNF4α Expression Leading to Impaired Human Hepatocyte Development and Function

“…We used wild-type (WT) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated 9 (Cas9) isogenic engineered hESCs harboring a clinically relevant mutation in the telomerase component DKC1 (DKC1_A353V mutation, which represents the most common mutation in patients with impaired telomere maintenance (21) ). While human induced pluripotent stem cells and hESCs have been used to study telomerase biochemistry in dyskeratosis congenita (DCs) (22)(23)(24) and mechanisms of hematopoietic failure in the setting of short telomeres, (21,25,26) , our data represent an initial attempt to use this technology to understand how telomere erosion leads to failure of hepatocyte development and function. We show that p53 activation following telomere erosion prevents expression of hepatocyte nuclear factor 4 alpha (HNF4α), a major transcription factor in hepatic cells, and leads to increased cellular proliferation, significantly impairing hepatocyte development and function in DC cells.…”