An enhanced H/ACA RNP assembly mechanism for human telomerase RNA

The integral telomerase RNA subunit templates the synthesis of telomeric repeats. The biological accumulation of human telomerase RNA (hTR) requires hTR H/ACA domain assembly with the same proteins that assemble on other human H/ACA RNAs. Despite this shared RNP composition, hTR accumulation is particularly sensitized to disruption by disease-linked H/ACA protein variants. We show that contrary to expectation, hTR-specific sequence requirements for biological accumulation do not act at an hTR-specific step of H/ACA RNP biogenesis; instead, they enhance hTR binding to the shared, chaperone-bound scaffold of H/ACA core proteins that mediates initial RNP assembly. We recapitulate physiological H/ACA RNP assembly with a preassembled NAF1/dyskerin/NOP10/NHP2 scaffold purified from cell extract and demonstrate that distributed sequence features of the hTR 3' hairpin synergize to improve scaffold binding. Our findings reveal that the hTR H/ACA domain is distinguished from other human H/ACA RNAs not by a distinct set of RNA-protein interactions but by an increased efficiency of RNP assembly. Our findings suggest a unifying mechanism for human telomerase deficiencies associated with H/ACA protein variants.     

Biological and biochemical functions of RNA in the tetrahymena telomerase holoenzyme

Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.