Fig. 6. Inhibition of oligoadenylation of hTR restores telomerase activity and telomere length in DKC1 mutant iPSCs.

a Northern blot analysis of hTR from WT_iPSC_F, R449G_iPSC_1 and R449G_iPSC_2 cells treated with RG7834, Cordycepin, or DMSO. A probe against 7SL RNA served as a loading control. b Total RNA prepared from WT_iPSC_F and R449G_iPSC_1 cells treated with RG7834, Cordycepin, or DMSO was subjected to qRT–PCR for oligoadenylated hTR. Bar graph of the mean fold change relative to DMSO-treated samples and normalized to GAPDH, ATP5b, and HPRT. Mean values were calculated from triplicate qRT–PCR experiments with three biological replicates, with bars representing standard error of the mean (s.e.m.). c Direct telomerase activity assay of cell lysates from WT_iPSC_F and R449G_iPSC_1 cells treated with RG7834 or cordycepin. 18-nucleotide telomeric primers (TTAGGG)3 were used. A ³²P end-labeled 18-nucleotide telomeric primers (TTAGGG)3 was added as loading control (LC). The numbers on the left (+4, +10, +16, +22, +28, etc.) indicate the number of nucleotides added to the telomeric-primer for each major band seen. d Telomere lengths determined by TRF analysis of gDNA prepared from WT_iPSC_F and R449G_iPSC_1 treated with RG7834, Cordycepin, or DMSO. e Schematic illustrating the dysfunction of DKC1_R449G in telomere maintenance.