Posttranscriptional manipulation of TERC reverses molecular hallmarks of telomere disease

Abstract

The telomerase RNA component (TERC) is a critical determinant of cellular self-renewal. Poly(A)-specific ribonuclease (PARN) is required for posttranscriptional maturation of TERC. PARN mutations lead to incomplete 3' end processing and increased destruction of nascent TERC RNA transcripts, resulting in telomerase deficiency and telomere diseases. Here, we determined that overexpression of TERC increased telomere length in PARN-deficient cells and hypothesized that decreasing posttranscriptional 3' oligo-adenylation of TERC would counteract the deleterious effects of PARN mutations. Inhibition of the noncanonical poly(A) polymerase PAP-associated domain-containing 5 (PAPD5) increased TERC levels in PARN-mutant patient cells. PAPD5 inhibition was also associated with increases in TERC stability, telomerase activity, and telomere elongation. Our results demonstrate that manipulating posttranscriptional regulatory pathways may be a potential strategy to reverse the molecular hallmarks of telomere disease.

Figure 1. TERC rescues telomere length in PARN-deficient cells.

(A) Telomere restriction fragment (TRF) length in HEK293 cells infected with shRNAs targeting luciferase (control) versus PARN. PD, population doubling. (B) TRF in PARN-knockdown HEK293 cells with lentivirus encoding TERC versus vector (ctrl). (C) TRF of immortalized, PARN-mutant patient fibroblasts with lentivirus encoding TERC versus vector.

Figure 2. PAPD5 inhibition restores TERC and telomerase activity in PARN-deficient cells.

(A) Strategy for 3′ RACE. (B) Agarose gel electrophoresis of 3′ RACE TERC products from HEK293 cells transduced with lentivirus encoding shRNA against PAPD5, PARN, or luciferase (ctrl). n = 4. (C) RACE products were subjected to deep sequencing and aligned to the TERC gene. Canonical TERC terminus is shown in red. Genomically encoded termini are in black, mature TERC with a single A (which may or may not be genomically encoded) is hatched, and oligo(A) additions of any length (n) are in solid blue. Total trimmed reads in parentheses. n = 2. *P ≤ 0.05; **P ≤ 0.01. (D) Oligo(An) species as a proportion of total reads in control versus PAPD5 knockdown cells. The average A-tail length in nucleotides is indicated. (E) Northern blot of TERC from HEK293 cells with shRNA against PAPD5 versus luciferase, followed by transfection with plasmids expressing PAPD5 versus EGFP cDNAs. Loading control, ethidium bromide staining of 18S rRNA. n = 3. (F) RACE amplicons from HEK293 cells with shRNA directed against PARN versus luciferase, followed by shRNA against PAPD5 or luciferase. n = 3. (G) Northern blot of TERC from HEK293 cells with shRNA against PARN versus luciferase, followed by shRNA against PAPD5 versus luciferase. n = 3. *P ≤ 0.05; ***P ≤ 0.001; NS: not significant. (H) Telomerase activity (TRAP) in PARN-deficient HEK293 cells after control versus PAPD5 knockdown, using 3-fold dilutions of input cell extract. IC, internal control amplification standard. n = 3.

Figure 3. PAPD5 inhibition rescues TERC maturation, telomerase activity, and telomere length in PARN-mutant patient cells.

(A) 3′ RACE TERC amplicons from normal (WT) versus PARN-mutant iPSCs after lentiviral shRNA directed against PAPD5 versus luciferase (ctrl). (B) Representative Northern blot of TERC RNA from WT versus PARN-mutant iPSCs after lentiviral shRNA directed against PAPD5 versus luciferase. (C) Relative TERC levels from Northern blots (B) are quantified relative to WT cells with control knockdown *P ≤ 0.05; NS: not significant. n = 3. (D) Representative Northern blot of TERC levels in WT and PARN-mutant iPSCs after lentiviral shRNA directed against PAPD5 versus luciferase at 0–4 hours following actinomycin treatment. TERC levels are normalized relative to time 0. (E) Graph of TERC decay rate. Dashed line reflects slope determined by simple linear regression. (F) TRAP assay in iPSCs after lentiviral shRNA against PAPD5 versus luciferase. n = 3. (G) TRF of WT and PARN-mutant patient iPSCs after lentiviral shRNA against PAPD5 versus luciferase over time in culture.