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. Author manuscript; available in PMC: 2016 Mar 14.
Published in final edited form as: Nat Genet. 2015 Oct 19;47(12):1482–1488. doi: 10.1038/ng.3423

Figure 6.

Figure 6

Ectopic expression of PARN rescues TERC maturation in PARN-deficient cells and shows that PARN is limiting for TERC biogenesis. (a) RNA blot of TERC RNA from HEK293 cells transduced with lentivirus encoding shRNA directed against PARN versus luciferase (control) and then rescued with lentivirus expressing PARN versus EGFP as a control. Ethidium bromide staining of 18S rRNA is used as a loading control. TERC levels normalized to those in cells with control knockdown and EGFP expression are shown. (b) 3′ RACE products from HEK293 cells subjected to shRNA-mediated knockdown of PARN f versus luciferase and rescued with PARN versus EGFP, separated by agarose gel electrophoresis. (c) RNA blot of TERC RNA from patient-derived fibroblasts transduced with lentivirus encoding PARN versus EGFP control. Normalized TERC levels are indicated. (d) 3′ RACE products from PARN-mutant patient-derived fibroblasts rescued with lentivirus expressing PARN versus EGFP. (e) Deep sequencing of 3′ RACE PCR products from HEK293 cells in which PARN is disrupted by shRNA and rescued by lentivirus expressing PARN or EGFP. Analysis was performed as in Figure 4. Statistical comparisons were between cells ectopically expressing PARN and those expressing EGFP. Significance is indicated: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; NS, not significant (black, mature TERC; blue, extended, oligo(A)n TERC forms). Error bars, s.d. (f) Deep sequencing of 3′ RACE PCR products from HEK293 control cells transduced with lentivirus overexpressing PARN versus EGFP. Statistical comparisons were performed as in e. Error bars, s.d.