Fig. 8: Telomerase activity and RNP assembly for hTR mutants.
From: Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq
a Activity assay of FLAG-purified HEK293T-derived telomerase measuring incorporation of 32P-dGTP into a telomeric DNA primer for the WT, M1, M2, and M3 hTR variant constructs. LC, labeled oligonucleotide loading controls. Experiment was repeated four times (n = 4) with similar results. b Quantification of telomerase activity. HEK293T- or HeLa-derived telomerase activity calculated by summing the total lane signal and correcting for differences in intensity of the loading controls. c Bar plot of fractional activity relative to WT was calculated from the slopes of the linear regression fits. d Western blots for hTERT (top), DKC1 (middle), and Gar1 (bottom) all performed after IP of FLAG-hTERT. Relative signal compared to wild type for two different loading amounts is shown below each lane. (Gar1 values are approximate due to accidental truncation of tops of bands.) Experiment was conducted two times (n = 2) with similar results. e Top, northern blot for hTR in HEK293T cell extracts, each hTR intensity normalized to U1 and U2 snRNA recovery controls and then to the WT hTR signal. Bottom, northern blot for hTR in anti-hTERT IP fractions, values normalized to WT. hTR migrates as a doublet when samples are heated and snap-cooled (right lanes) because the 7 M urea gel is not completely denaturing. Experiment was conducted four times (n = 4) with similar results.
