Specificity and stoichiometry of subunit interactions in the human telomerase holoenzyme assembled in vivo

Abstract

The H/ACA motif of human telomerase RNA (hTR) directs specific pathways of endogenous telomerase holoenzyme assembly, function, and regulation. Similarities between hTR and other H/ACA RNAs have been established, but differences have not been explored even though unique features of hTR H/ACA RNP assembly give rise to telomerase deficiency in human disease. Here, we define hTR H/ACA RNA and RNP architecture using RNA accumulation, RNP affinity purification, and primer extension activity assays. First, we evaluate alternative folding models for the hTR H/ACA motif 5' hairpin. Second, we demonstrate an unanticipated and surprisingly general asymmetry of 5' and 3' hairpin requirements for H/ACA RNA accumulation. Third, we establish that hTR assembles not one but two sets of all four of the H/ACA RNP core proteins, dyskerin, NOP10, NHP2, and GAR1. Fourth, we address a difference in predicted specificities of hTR association with the holoenzyme subunit WDR79/TCAB1. Together, these results complete the analysis of hTR elements required for active RNP biogenesis and define the interaction specificities and stoichiometries of all functionally essential human telomerase holoenzyme subunits. This study uncovers unexpected similarities but also differences between telomerase and other H/ACA RNPs that allow a unique specificity of telomerase biogenesis and regulation.

FIG. 1.

The hTR H/ACA motif 5′ hairpin is noncanonical and has few structural requirements for hTR accumulation. (A) Secondary structure and primary sequence motifs of hTR are illustrated. (B) At left, the secondary structure of the originally proposed hTR H/ACA motif 5′ hairpin is shown with boxes around 4 or 2 nt on each side of the putative stem pairings tested by substitution. At right is shown an alternative, more canonical secondary structure model for the H/ACA motif 5′ hairpin based on a 5′ domain boundary at position 225. P4, P4.1, and P4.2 are paired elements predicted by phylogenetic comparison; P4a (alt.) and P4b (alt.) are alternative pairings of the residues involved in P4 and P4.1. (C) Total RNA from transfected 293T cells was examined by blot hybridization. Empty-vector lanes provide a background control for detection of endogenous hTR compared to recombinant wild-type (WT) hTR or the hTR variants indicated. Full-length hTR, the 5′ processed hTR H/ACA domain, and the transfection control (TC) were detected on the same blot. In the boxed and numbered regions of hTR secondary structure taken from panel B, the substituted sequences tested in panel C are specified. L and R indicate left and right side of the stem pairings as illustrated. (D) Total RNA from transfected 293T cells was examined by blot hybridization. At left is shown the hTR H/ACA motif from the most recent secondary structure model based on phylogenetic comparison. Pocket sequences are highlighted in bold. L del, R del, L+R del indicate deletion of the left, right, or combined sides of the pocket, respectively. Pairings forced in the hTR variant of lane 6 are shown with connecting lines; all other pocket residues were deleted. The five right-side pocket residues retained in the hTR variants of lanes 7 and 8 are boxed; the other eight right-side pocket residues were deleted (R8 del).

FIG. 2.

Holoenzyme catalytic activity does not require any specific paired region of the 5′ hairpin stem or the 5′ hairpin pocket. Direct primer extension activity assays were performed using extracts of VA13+TERT cells transfected to express the indicated hTR variants. Levels of hTR in the extracts are shown by blot hybridization; note that the hTR variant of lane 8 was slightly underaccumulated in this set.

FIG. 3.

A minimal 5′ hairpin stem is sufficient for accumulation of hTR and snoRNAs. Total RNA from transfected 293T cells was examined by blot hybridization. At left are shown secondary structures of the hTR H/ACA domain phylogenetic model (A), the human snoRNA U64 (B), and the human snoRNA ACA28 (C). Positions of internal deletion are indicated: Δ1, deletion of H/ACA motif upper stem; Δ2, deletion of the upper stem and pocket; Δ3, deletion of the entire stem/pocket/stem hairpin. Only hTR Δ2 and the snoRNA 5′ stem cap (SC) deletions insert a GAAA tetraloop in place of the deleted sequence. Human snoRNA pocket sequences are highlighted in bold. L del, R del, L+R del indicate deletion of the left, right, or combined left and right sides of the pocket, respectively.

FIG. 4.

Two subunits of dyskerin, NHP2, and GAR1 assemble on each molecule of hTR. (A) Schematic of the tandem affinity purification strategy for discriminating subunit stoichiometry. (B) Extracts from 293T cells transfected to express a protein with the tag(s) indicated (Z, F, +, and ZF, where + indicates coexpression of the Z and F tags), wild-type hTR, and the hTR-U64 chimera were subjected to tandem steps of affinity purification. Input cell extracts (2%) and final purified RNP elutions (100%) were supplemented with a recombinant RNA recovery control (RC) prior to RNA extraction. Input and purified samples for the GAR1 panel were from a separate set of transfections and purifications.

FIG. 5.

Two subunits of dyskerin, NHP2, and GAR1 assemble on hTR variants lacking consensus elements of the 5′ hairpin. Extracts from 293T cells transfected to express a protein with the tag(s) indicated (Z, F, +, and ZF), hTR-U64, and one of two hTR variants (5′ pocket L+R del from Fig. 1D or Δ2 from Fig. 3A) were subjected to tandem steps of affinity purification. RNAs in the input and purified material were analyzed as described in the legend of Fig. 4. Note that hTR Δ2 migrated with different mobilities depending on sample heating. The input and purified samples of any given protein were analyzed in parallel.

FIG. 6.

Association of WDR79/TCAB1 is influenced by the hTR CAB box. (A) Extracts from 293T cells transfected to express hTR and WDR79/TCAB1 with the tag(s) indicated (Z, F, +, and ZF) were subjected to tandem steps of affinity purification. RNAs in the input and purified material were analyzed as described in the legend of Fig. 4. (B) Extracts of VA13+TERT cells transfected to express either Z-tagged WDR79/TCAB1 or empty vector and either wild-type (WT) hTR or the G414C CAB box mutant (Mut) were used for single-step affinity purification. RNAs in the input and purified material were analyzed as described above.

FIG. 7.

Interaction specificity and stoichiometry of telomerase holoenzyme proteins. Initial recruitment of H/ACA proteins is proposed to involve an association of dyskerin, NOP10, and NHP2 with the hTR 3′ hairpin, scaffolded by extensive protein-RNA interactions. Either sequentially (as shown) or in a concerted manner, there is assembly of a second H/ACA protein heterotrimer. Assembly of the second set of H/ACA proteins is proposed to have a reduced requirement for RNA interaction surface due to the formation of cross-hairpin protein-protein interaction(s). Following hTR release from the site of transcription, an exchange of biogenesis factors for GAR1 yields the mature telomerase RNP with two full sets of all four H/ACA RNP proteins. Motifs of hTR not required for stable RNP biogenesis can associate independently with TERT and WDR79/TCAB1, both of which are likely substoichiometric in the overall population of endogenous telomerase RNP.