A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

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.     

The 5' guanosine tracts of human telomerase RNA are recognized by the G-quadruplex binding domain of the RNA helicase DHX36 and function to increase RNA accumulation

Telomerase promotes telomere maintenance by copying a template within its integral RNA subunit to elongate chromosome ends with new telomeric repeats. Motifs have been defined within the telomerase RNA that contribute to mature RNA accumulation, holoenzyme catalytic activity, or enzyme recruitment to telomeres. Here, we describe a motif of human telomerase RNA (hTR), not previously characterized in a cellular context, comprised of several guanosine tracts near the RNA 5' end. These guanosine tracts together are recognized by the DEXH box RNA helicase DHX36. The helicase domain of DHX36 does not mediate hTR binding; instead, hTR interacts with the N-terminal accessory domain of DHX36 known to bind specifically to the parallel-strand G-quadruplex substrates resolved by the helicase domain. The steady-state level of DHX36-hTR interaction is low, but hTR guanosine tract substitutions substantially reduce mature hTR accumulation and thereby reduce telomere maintenance. These findings suggest that G-quadruplex formation in the hTR precursor improves the escape of immature RNP from degradation, but subsequently the G-quadruplex may be resolved in favor of a longer terminal stem. We conclude that G-quadruplex formation within hTR can stimulate telomerase-mediated telomere maintenance.