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

Nature Structural & Molecular Biology volume 23, pages 286–292 (2016)Cite this article

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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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Figure 1: Lack of dyskerin binding reduces hTR levels through two different RNA decay mechanisms.
Figure 2: PARN knockdown reduces hTR levels, owing to competing activity of EXOSC10.
Figure 3: Rescue of telomerase activity in dyskerin- or PARN-depleted HeLa cells by coknockdown of competing nucleases.
Figure 4: Mislocalization of hTR in dyskerin- or PARN-knockdown HeLa cells can be corrected by knockdown of competing nuclease.
Figure 5: Mutant hTR localization to Cajal bodies can be rescued by knockdown of RNA decay pathways.
Figure 6

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Acknowledgements

We would like to thank A. Zaug for his assistance with the telomerase direct activity assays; A. Webb for help with the illustrations and figures in this manuscript; S. Spencer (University of Colorado Boulder), J. Lingner (Institut Suisse de Recherche Expérimentale sur le Cancer) and S.B. Cohen (Children's Medical Research Institute) for providing materials; and members of the Parker and Cech laboratories for their comments and suggestions. J.C.S. is supported as a Merck fellow of the Damon Runyon Cancer Research Foundation (DRG-2169-13). This work was supported by US National Institutes of Health grants R01 GM45443 (R.P.) and GM099705 (T.R.C.). T.R.C. and R.P. are supported as investigators of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Chemistry & Biochemistry, University of Colorado, Boulder, Colorado, USA

    Siddharth Shukla, Jens C Schmidt, Katherine C Goldfarb, Thomas R Cech & Roy Parker

  2. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Thomas R Cech & Roy Parker

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  1. Siddharth Shukla
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  2. Jens C Schmidt
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Contributions

S.S., J.C.S., T.R.C. and R.P. conceptualized and designed the experiments. S.S. performed the experiments. J.C.S. performed the Pol II ChIP for hTR. K.C.G. and S.S. analyzed the hTR 3′-end sequencing reads. S.S., J.C.S., T.R.C. and R.P. analyzed and interpreted data. S.S., J.C.S., T.R.C. and R.P. wrote the manuscript.

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Correspondence to Roy Parker.

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Competing interests

T.R.C. is on the board of directors of Merck, Inc. The other authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quality-control pathways for A377G and C408G mutant hTR are conserved in U2OS cells.

Knockdown of specific nucleases rescues levels of A377G and C408G hTR in U2OS cells. a, Western blot for knockdown of various nucleases in U2OS cells. b, Northern blot for A377G hTR levels upon knockdown of DCP2, EXOSC10 or XRN1 (mean+/-s.d., n=4 independent experiments). c, Northern blot for C408G hTR levels upon PAPD5 knockdown with or without a co-knockdown of DCP2 or XRN1 (mean+/-s.d., n=3 independent experiments). d, Northern blot for C408G hTR upon co-knockdown of EXOSC10 and PAPD5 (mean+/-s.d., n=4 independent experiments). e, Northern blot for hTR upon knockdown of various decapping enzymes in U2OS cells (mean+/-s.d., n=4 independent experiments). f, Normalized Pol II occupancy by ChIP for the wild-type and C408G hTR plasmid-borne copy in U2OS cells (wild-type arbitrarily set to 1 and C408G normalized to wild-type).

Supplementary Figure 2 hTR 3′ mature end exhibits longer A tails after PARN knockdown.

Length distribution of oligo(A) tails at the mature 3’ end of hTR under different transfection conditions. Relative amount of each two or more A-containing reads was calculated to the total number of two or more A’s containing hTR reads under each transfection condition (P<0.05 by one tail unpaired Student’s t test).

Supplementary Figure 3 Mutant hTR localizes to cyTER bodies in U2OS cells.

Mutant A377G or C408G hTR is mislocalized to the cytoplasm in cyTER bodies in U2OS cells. a, Cytoplasmic localization of A377G or C408G RNA (white arrowheads) by FISH in U2OS cells. The nucleus is labeled with an antibody that recognizes either of the nuclear envelope components lamin A or lamin C (Scale bar 5 μm). White numbers in Merge, % of cells showing hTR localization to Cajal bodies (mean+/-s.d., n=4 independent experiments). b, Cytoplasmic localization of C408G hTR by FISH (nucleus outlined with lamin A/C staining, cell outlined with pan cadherin staining) (Scale bar 5 μm). White numbers in Merge, % of cells showing hTR cytoplasmic localization (mean+/-s.d., n=4 independent experiments).

Supplementary Figure 4 FISH signal for WT and mutant hTR is specific and does not localize to P bodies or U bodies.

cyTER bodies are novel sites of hTR storage. a, Subcellular localization of WT or C408G hTR (white arrowhead) by FISH using different combinations of Alexa Fluor labeled probes (Scale bar 5 μm). b, Subcellular localization of C408G RNA puncta (white arrowhead) in U2OS cells. Other foci stained were U-bodies or Gems (SMN) or P-bodies (DCP1) (Scale bar 10μm).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 643 kb)

Supplementary Data Set 1

Uncropped blots and autoradiograph images (PDF 11407 kb)

Supplementary Table 1

Raw data for hTR 3′ end sequencing under different conditions (XLSX 18 kb)

Supplementary Table 2

Compiled number of reads for hTR 3′ end sequencing (XLSX 18 kb)

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Shukla, S., Schmidt, J., Goldfarb, K. et al. Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects. Nat Struct Mol Biol 23, 286–292 (2016). https://doi.org/10.1038/nsmb.3184

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