Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Human telomerase RNA can accumulate without H/ACA RNP biogenesis.
(A,B) Diagrams of hTR and hTRmin secondary structure and bound proteins. (C) Parts list for a cellular minimal telomerase RNA. Components are presented in 5’ to 3’ order. (D,E) Northern blot assay …
Figure 1—figure supplement 1
Cellular assembly of hTRmin telomerase.
(A) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr …
Figure 2 with 1 supplement
An hTRmin RNP can functionally substitute for hTR.
(A) Schematic of Cas9-mediated disruption of the hTR locus with a PURO selection cassette. (B) Northern blot and hotTRAP assays of hTR KO#1 and KO#2 clonal HCT116 cell lines. An internal control …
Figure 2—figure supplement 1
Generation of cell lines expressing hTRmin telomerase.
(A) Schematic and assay results for PCR detection of hTR locus KO. (B) Telomerase activity comparison by hotTRAP for the hTR KO cell lines with TERT OE alone or in combination with hTRmin or …
Figure 3 with 1 supplement
Telomerase with hTRmin supports stable telomere length maintenance.
(A) Southern blot detection of telomere restriction fragment lengths (TRF) for HCT116 cell lines after release from selection. The telomerase-negative cell line TRFs were analyzed before cultures …
Figure 3—figure supplement 1
Characterization of telomere length maintenance and telomerase activity levels in hTRmin telomerase cell lines.
(A) Northern blot and TRF timecourse of HCT116 hTR KO#2 cell line rescue by transgene hTR. (B) Time course of HCT116 TRF after introduction of a transgene for TERT OE alone or with LhTRmin or hTRmin;…
Figure 4 with 1 supplement
TCAB1 and Cajal bodies are not required for telomere maintenance by hTRmin telomerase.
(A) Schematic of Cas9-mediated disruption of TCAB1 and Coilin (COIL) loci with a HYGRO selection cassette. (B) Immunoblot and QTRAP analysis of the polyclonal populations of LhTRmin or hTRmin TERT …
Figure 4—figure supplement 1
Characterization of HCT116 hTRmin with TERT OE cell lines with TCAB1 KO or Coilin KO.
(A) Immunopurification from HCT116 hTR KO cell lines expressing LhTRmin with TERT OE or hTRmin with TERT OE. Protein A/G beads were coupled with anti-TCAB1 antibody and used to immunopurify TCAB1 …
Figure 5 with 3 supplements
TCAB1 and Cajal bodies are not essential for telomere maintenance by endogenous telomerase.
(A) Immunoblot analysis of HCT116 TCAB1 KO and COIL KO clonal cell lines. (B) Northern blot for hTR in HCT116 TCAB1 KO and COIL KO clonal cell lines. (C) Lack of hotTRAP telomerase activity …
Figure 5—figure supplement 1
Characterization of HCT116 TERT KO, TCAB1 KO, and COIL KO cell lines with endogenous hTR.
(A) PCR genotyping of HCT116 TERT KO clonal cell lines. (B) Immunopurification of TCAB1 complexes from parental, TCAB1 KO, and COIL KO HCT116 cell lines followed by hotTRAP. (C) HotTRAP of HCT116 …
Figure 5—figure supplement 2
Characterization of hESC TCAB1 KO and COIL KO lines with endogenous hTR.
(A) PCR genotyping of hESC TCAB1 KO and COIL KO clonal cell lines. Wild-type refers to an hESC clonal cell line subjected to Cas9 electroporation but retaining a wild-type genotype. (B) HotTRAP of …
Figure 5—figure supplement 3
Additional controls for TCAB1 loss-of-function in TCAB1 KO cell lines.
(A) Immunoblot and QTRAP analysis of HCT116 parental and TCAB1 KO cell lines expressing F-tagged TCAB1 or GFP from transgenes at AAVS1. QTRAP values were normalized to the GFP cell line signal (n = 3…
Figure 6
Cajal bodies promote telomere elongation upon increased telomerase expression level.
(A) Immunoblot, northern blot, and QTRAP characterization of TCAB1 KO and COIL KO HCT116 cells overexpressing hTR and TERT at AAVS1 (HiT). QTRAP values were normalized to parental HCT116 (n = 3). (B)…
Figure 7
Multiple traffic pathways for human telomerase biogenesis and action at telomeres.
At left, hTRmin telomerase assembles and acts at telomeres without supplemental trafficking instructions. At right, endogenous hTR and TERT are trafficked for their assembly and for telomerase …
