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. 2016 Apr;23(4):286-92.
doi: 10.1038/nsmb.3184. Epub 2016 Mar 7.

Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects

Siddharth Shukla  1 , Jens C Schmidt  1 , Katherine C Goldfarb  1 , Thomas R Cech  1   2 , Roy Parker  1   2
Affiliations

Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects

Siddharth Shukla et al. Nat Struct Mol Biol. 2016 Apr.

Abstract

Mutations in the human telomerase RNA component (hTR), the telomerase ribonucleoprotein component dyskerin (DKC1) and the poly(A) RNase (PARN) can lead to reduced levels of hTR and to dyskeratosis congenita (DC). However, the enzymes and mechanisms responsible for hTR degradation are unknown. We demonstrate that defects in dyskerin binding lead to hTR degradation by PAPD5-mediated oligoadenylation, which promotes 3'-to-5' degradation by EXOSC10, as well as decapping and 5'-to-3' decay by the cytoplasmic DCP2 and XRN1 enzymes. PARN increased hTR levels by deadenylating hTR, thereby limiting its degradation by EXOSC10. Telomerase activity and proper hTR localization in dyskerin- or PARN-deficient cells were rescued by knockdown of DCP2 and/or EXOSC10. Prevention of hTR RNA decay also led to a rescue of localization of DC-associated hTR mutants. These results suggest that inhibition of RNA decay pathways might be a useful therapy for some telomere pathologies.

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Conflict of interest statement

The other authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Lack of dyskerin binding reduces hTR levels by two different RNA decay mechanisms. a, Western blot for DKC1 knockdown and northern blot for hTR in HeLa cells (mean +/−s.d., n=5 independent experiments). Scr, scrambled negative control siRNA. b, c, Representative northern blot for hTR upon DKC1 knockdown and rescue in HeLa cells (mean+/−s.d., n=4 independent experiments. d,e, Representative northern blot for hTR upon Actinomycin D shutoff in dyskerin knockdown and rescue in HeLa cells. Measurement of hTR decay rate in dyskerin knockdown HeLa cells. Error bars, s.d. (n=3 independent experiments). f, Northern blot showing hTR upon DKC1 knockdown with or without PAPD5 knockdown (mean+/−s.d., n=3 independent experiments). g, Northern blot for wild type (WT) disease-causing hTR mutants in U2OS cells (mean+/−s.d., n=3 independent experiments). h, Northern blot for C408G hTR RNA upon DCP2, EXOSC10 or XRN1 knockdown (mean+/−s.d., n=4 independent experiments).
Figure 2
Figure 2
PARN knockdown reduces hTR levels due to competing activity of EXOSC10. a, Model for competition between PARN and EXOSC10 for access to adenylated hTR 3’ end mediated by PAPD5. b, Northern blot for hTR upon PARN knockdown and rescue by EXOSC10 or PAPD5 co-knockdown (mean+/−s.d., n=6 independent experiments). c, d, Representative northern blot for Actinomycin D shutoff in PARN knockdown and rescue in HeLa cells. Measurement of hTR decay rate in PARN knockdown HeLa cells. Error bars, s.d. (n=4 independent experiments). e, Relative abundance of oligoadenylated reads at the mature hTR 3’ end with different components knocked down. Reads were normalized to the total number of mature end-containing reads under each condition.P<0.001 by two tailed Student’s t test for total number of reads in each condition(Supplementary Table 1).
Figure 3
Figure 3
Rescue of telomerase activity in dyskerin- or PARN-depleted HeLa cells by co-knockdown of competing nucleases. a, Autoradiograph for telomerase activity assay in HeLa DKC1 knockdown and rescue cells. LC1 and LC2, oligonucleotide loading controls.1–6, telomeric repeats added to the telomeric primer. b, Telomerase activity relative to the Scr control sample. Error bars, s.d. (n=5 independent experiments). ** P<0.01 by one-tailed unpaired Student’s t test. c, d, Autoradiograph for telomerase activity assay in HeLa PARN knockdown and rescue cells. Labeling of blot as in a. Error bars, s.d. (n=5 independent experiments), * P<0.05, ** P<0.01 by one-tailed unpaired Student’s t test.
Figure 4
Figure 4
Mislocalization of hTR in dyskerin or PARN knockdown HeLa cells can be corrected by knockdown of competing nuclease. a, Subcellular localization of hTR by FISH in HeLa cells upon dyskerin knockdown (white arrowheads), and the localization to Cajal bodies upon rescue by DCP2 or DCP2 and EXOSC10 co-knockdown (light brown arrowheads) (Scale bar 5 μm). White numbers in Merge, % of cells showing hTR localization to CBs (mean+/−s.d., n=3 independent experiments). b, Subcellular localization of hTR by FISH to the cytoplasm or to CBs upon PARN knockdown and rescue, respectively (light brown arrowheads) (Scale bar 5 μm). White numbers in Merge, % of cells showing hTR localization to CBs (mean+/−s.d., n=4 independent experiments).
Figure 5
Figure 5
Mutant hTR localization to Cajal bodies can be rescued by knockdown of RNA decay pathways. a, WT hTR localization to neo-Cajal bodies at telomeres (labeled with TRF2) by FISH in cells overexpressing hTERT (Scale bar 5μm). White numbers in Merge, % of cells showing hTR localization to CBs (mean+/−s.d., n=4 independent experiments). b, Nuclear localization of A377G mutant hTR to Cajal bodies by FISH (light brown arrowheads) in hTERT co-transfected U2OS cells upon knockdown of nucleases (Scale bar 5μm). White numbers in Merge, % of cells showing hTR localization to CBs (mean+/−s.d., n=3 independent experiments).
Figure 6
Figure 6
Model for hTR biogenesis, depicting competition between hTR assembly with H/ACA snoRNP proteins, 3’ end processing by PAPD5 and PARN and degradation by EXOSC10, and cytoplasmic export and degradation by DCP2 and XRN1.
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References

    1. Blackburn E, Collins K. Telomerase: An RNP Enzyme Synthesizes DNA. Cold Spring Harb Perspect Biol. 2011;3:a003558. - PMC - PubMed
    1. Armanios M, Blackburn E. The telomere syndromes. Nat Rev Genet. 2012;13:693–704. - PMC - PubMed
    1. Heiss NS, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet. 1998;19:32–8. - PubMed
    1. Vulliamy T, et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature. 2001;413:432–5. - PubMed
    1. Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature. 1999;402:551–5. - PubMed

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