Identification of TCAB1 as a dyskerin- and telomerase-interacting protein.
(A) Dual affinity-purified dyskerin complexes fractionated by SDS-PAGE and silver stained. (B) Unique and total peptides corresponding to dyskerin-associated proteins identified by nanoLC MS/MS (C) Flag-dyskerin interactions with endogenous TCAB1 and telomerase components and (D) Flag-TCAB1 and Flag-NAF1 interactions with endogenous telomerase components. Immunoprecipitation-western blot using extracts from Flag-dyskerin^+shRNA cells, Flag-TCAB1^+shRNA cells or Flag-NAF1^+shRNA cells. IB, immunoblot. NB, northern blot. parental, HeLa cells. +, treatment of extracts with RNase A during immunoprecipitation. Recovery control, exogenous RNA spiked into samples after IP to control for differential RNA recovery.

TCAB1 is enriched in Cajal bodies and associates specifically with small Cajal body RNAs (scaRNAs).
(A) HA-TCAB1 colocalizes with the Cajal body marker p80-coilin. Immunofluorescence (IF) using anti-HA (rat) and anti-p80-coilin (mouse) antibodies on fixed HeLa cells stably overexpressing HA-TCAB1. (B) Co-localization of endogenous TCAB1 with p80-coilin. IF using anti-TCAB1 (rabbit) and anti-p80-coilin antibodies (mouse) on fixed HeLa cells. (C) Flag-TCAB1 associates specifically with scaRNAs by immunoprecipitation-northern blot. Cell extract from parental HeLa cells, Flag-NAF1^+shRNA cells, Flag-TCAB1^+shRNA cells or Flag-dyskerin^+shRNA cells was incubated with Flag antibody beads. RNA was isolated from extracts before (input) and after (depleted) antibody treatment. Two μg total RNA was fractionated by urea-PAGE (lanes 1-8), representing 2% of the input. RNAs from IPs, lanes 9-12 (D) Endogenous TCAB1 associates specifically with scaRNAs. NAF1 antibodies, TCAB1 antibodies or IgG control were used in assays at the same scale as in (C). RNAs from input and depleted extracts, lanes 1-5. RNAs from IPs, lanes 6-8. Recovery control, exogenous RNA spiked into samples after IP to control for differential RNA recovery.

TCAB1 is a component of active telomerase.
(A) Flag-TCAB1 or Flag-dyskerin immunoprecipitation (IP) quantitatively co-depletes telomerase activity and TERC from extracts. TRAP assays were performed on extracts before and after IP (lanes 1-8) and on each immunoprecipitated complex (lanes 10-13). Depletion of tagged proteins and depletion of endogenous TERC on extracts before and after IP (lanes 14-21). IP of tagged proteins and associated telomerase components (lanes 22-25). U1 splicing RNA, negative control. (B) IP of endogenous TCAB1 co-depletes telomerase activity and TERC. TRAP assays were performed on extracts before and after each IP (lanes 1-4) and on the IP (lanes 6-8). Depletion of endogenous NAF1 or endogenous TCAB1 and depletion of TERC (lanes 9-13). Association of NAF1 and TCAB1 with telomerase components (lanes 14-16). Recovery control, exogenous RNA spiked into samples after IP to control for differential RNA recovery.

TCAB1 is essential for TERC localization to Cajal bodies and for telomere synthesis by telomerase.
(A) HeLa cells were transduced with retroviruses expressing independent shRNA sequences targeting the indicated proteins or with empty vector control. Telomerase activity was measured by TRAP assay. (B) TERC colocalization with p80-coilin was determined by RNA FISH for TERC (green) and immunofluorescence for p80-coilin (red)(top). Cells synchronized in S phase were assayed for TERC by RNA FISH (green) and for telomeres with TRF2 antibodies (red) to assess trafficking of TERC to telomeres (bottom). (C) Quantification of data in (B). Top: cells in which TERC colocalized with p80-coilin (+) versus cells in which TERC was not detected in Cajal bodies (-), p<0.0001 by Fisher’s exact test. Bottom: cells in which TERC colocalized with telomeres (+) versus cells in which TERC was not detected at telomeres (-), p<0.0001 by Fisher’s exact test. (D) Telomere lengths were measured by TRF Southern blot in HTC75 cells overexpressing wild-type TERC (lanes 1-8) or mutant TERC-m1 (lanes 9-12). Cells overexpressing wild-type TERC were transduced with shRNA retroviruses targeting TCAB1 or with empty vector. (E) Effect of TCAB1 depletion on endogenous telomerase was assessed by TRF Southern blot in parental HTC75 cells transduced with empty vector or retroviruses expressing independent TCAB1 shRNAs. Cells were counted at each passage and population doublings are indicated (F) Model for TCAB1 function in the telomere synthesis pathway.