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. 2016 Aug 30;113(35):E5125-34.
doi: 10.1073/pnas.1607411113. Epub 2016 Aug 16.

Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

Yaqiang Wang  1 , Joseph D Yesselman  2 , Qi Zhang  3 , Mijeong Kang  4 , Juli Feigon  5
Affiliations

Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

Yaqiang Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Telomerase is an RNA-protein complex that includes a unique reverse transcriptase that catalyzes the addition of single-stranded telomere DNA repeats onto the 3' ends of linear chromosomes using an integral telomerase RNA (TR) template. Vertebrate TR contains the template/pseudoknot (t/PK) and CR4/5 domains required for telomerase activity in vitro. All vertebrate pseudoknots include two subdomains: P2ab (helices P2a and P2b with a 5/6-nt internal loop) and the minimal pseudoknot (P2b-P3 and associated loops). A helical extension of P2a, P2a.1, is specific to mammalian TR. Using NMR, we investigated the structures of the full-length TR pseudoknot and isolated subdomains in Oryzias latipes (Japanese medaka fish), which has the smallest vertebrate TR identified to date. We determined the solution NMR structure and studied the dynamics of medaka P2ab, and identified all base pairs and tertiary interactions in the minimal pseudoknot. Despite differences in length and sequence, the structure of medaka P2ab is more similar to human P2ab than predicted, and the medaka minimal pseudoknot has the same tertiary interactions as the human pseudoknot. Significantly, although P2a.1 is not predicted to form in teleost fish, we find that it forms in the full-length pseudoknot via an unexpected hairpin. Model structures of the subdomains are combined to generate a model of t/PK. These results provide evidence that the architecture for the vertebrate t/PK is conserved from teleost fish to human. The organization of the t/PK on telomerase reverse transcriptase for medaka and human is modeled based on the cryoEM structure of Tetrahymena telomerase, providing insight into function.

Keywords: NMR; RNA structure; RNP; TERT; medaka.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Medaka and human TR. (A) The secondary structure of the hFL-TR pseudoknot. The red letters indicate nucleotides with >80% sequence conservation among vertebrates. (B) Predicted secondary structure of mdTR containing the t/PK, CR4/5, and H/ACA domains. The FL-PK sequence and base pairs predicted by phylogenetic comparative analysis are shown in the box on the left. The 100%-conserved nucleotides identified in the five teleost fish TR are highlighted in red. TERT (gray ellipse) interacts with the t/PK and CR4/5 domains. The stems and loops are labeled and colored individually: P2a.1, purple; P2a, orange; P2b, red; P3, blue; J2a/b, green; J2a/3, black; and J2b/3, cyan.
Fig. 2.
Fig. 2.
MdTR minimal (P2b–P3) pseudoknot. (A) Sequence and secondary structure of the minimal pseudoknot construct mdPK used in the solution NMR study. (B) Imino region of HNN-COSY (Upper) aligned with imino NOESY spectrum of mdPK (Lower). NOE cross-peaks of base pairs are connected and colored as in Fig. 1. (C) Stick representation of the G62–C73–G132 base triple. Secondary structure elements are colored as in Fig. 1.
Fig. S1.
Fig. S1.
TOCSY spectra of the minimal mdPK constructs. (A) mdPK-0. (B) mdPK. (C) mdPKΔA131. (D) mdPKA131U. Spectra were acquired at 800 MHz, 10 °C. Sequence and secondary structures of each construct are shown in the upper left corners. Secondary structure elements are colored as in Fig. 1.
Fig. S2.
Fig. S2.
1D imino proton of the minimal mdPK constructs at 800 MHz and 10 °C. (A) mdPK-0. (B) mdPK. (C) mdPKΔA131. (D) mdPKA131U. The blue and cyan dashed lines connect imino resonances (62, 74) with different chemical shifts in different constructs. Sequence and secondary structures of each construct are shown at right. Secondary structure elements are colored as in Fig. 1.
Fig. S3.
Fig. S3.
Comparison of spectra of mdPKΔA131 and mdPK. (A) The imino region of the H2O NOESY spectrum of mdPKΔA131. (B) Superimposition of the imino region of the H2O NOESY spectra of mdPK (black) and mdPKΔA131 (red). (C) Region of the H2O NOESY spectrum of mdPK showing cross-peaks of G62 imino.
Fig. 3.
Fig. 3.
Comparison of minimal mdPK and hPK (PDB ID code 2K95) structures. Secondary structure elements are P2b (red), P3 (blue), J2a/3 (green), and J2b/3 (gold).
Fig. 4.
Fig. 4.
Telomerase activity assays of mdTR mutants. (A) Secondary structure of FL-PK with nucleotide substitutions indicated by arrows. (B) Effect of mdP2a1a, mdP2ab, and mdPK mutations (from left to right) on telomerase activity. Medaka TERT synthesized in RRL was assembled with full-length WT (first lane for each subgroup) or mutant mdTR. RC, recovery control. (C) Plot of the activity of each mutant relative to that of WT mdTR. The error bars indicate the difference or SD calculated from two or three independent assay reactions, respectively. Secondary structure elements and corresponding labels and bars in the plot are colored as in Fig. 1.
Fig. 5.
Fig. 5.
Solution structure of the mdP2ab (P2a–J2a/b–P2b). (A) Sequence and secondary structure of predicted (Left) and observed (Right) mdP2ab. (B) Superposition of the 20 lowest-energy structures over all heavy atoms and space-filling rendering of the lowest-energy structure of P2ab. (C) Comparison of the lowest-energy structure of mdP2ab (Left) and hP2ab (PDB ID code 2L3E) (Right). In all panels, P2a is gold, J2a/b is green, P2b is red, and the UUCG tetraloop is gray.
Fig. S4.
Fig. S4.
Normalized resonance intensities of mdP2ab from non–constant-time 1H-13C HSQC experiments. The resonance intensities of each type of C–H spin were normalized against the lowest intensity from the helix to the reference value of 0.1. The resonances and nucleotide labels are colored by subdomain as in Fig. 5.
Fig. 6.
Fig. 6.
The FL-PK. (A–D) 1D imino proton spectra of mdP3 (A), mdP2b (B), mdPK (C), and mdFL-PK (D). (E) Imino proton region of the NOESY spectrum of mdFL-PK. (Inset) Sequence and secondary structure. Secondary structure elements are colored as in Fig. 1.
Fig. S5.
Fig. S5.
Superimposition of the imino region of H2O NOESY spectra of mdFL-PK (black), mdPK (blue), and mdP2a1a (purple). The assignments of mdFL-PK are labeled along the diagonal using the color scheme in Fig. 1.
Fig. S6.
Fig. S6.
Comparison of the imino regions of H2O NOESY spectra of mdP2b, mdP3, and mdFL-PK. (A) mdP2b. (B) mdP3. (C) Superimposition of mdFL-PK (black) and mdP2b (red) spectra. The assignments of mdFL-PK are labeled along the diagonal using the color scheme in Fig. 1. Resonances from mdP2b are labeled in red to demonstrate the presence of a small amount of the P2b hairpin in mdFL-PK.
Fig. 7.
Fig. 7.
MdTR P2a1a. (A) Proposed secondary structure of mdP2a1a, composed of a P2a.1 hairpin and part of the P2a stem capped with a UUCG tetraloop. (B) The ensemble of the 20 lowest-energy structures. (C) The three lowest-energy models for the P2a.1 hairpin. (D) The imino proton region of the NOESY spectrum of P2a1a.
Fig. S7.
Fig. S7.
Regions of the NOESY spectrum of mdP2a1a showing aromatic, H5, and H1′ cross-peaks. (A) The cross-peak between C94H6 and G95H8 indicates that P2a.1 stacks on P2a. (B) The cross-peaks of C35H5–U110H5 and C36H5–U110H5 indicate that 3′ UUGA (nucleotides 109–112) interacts with P2a.
Fig. S8.
Fig. S8.
Proposed secondary structure of teleost fish TR t/PK with the P2a.1 hairpin. At right is comparison of the estimated length of the J2a/3 loop (in nucleotides) vs. P2ab (in base pairs). Non-Watson--Crick pairs, but not bulge nucleotides, are included in the base-pair count.
Fig. S9.
Fig. S9.
MdTR P2a.1 mutant hairpin (GUU95–97CGA, AGC106–108UUG) with 5' and 3' stem sequence swapped. (A) The three lowest-energy models from the ∼2,500 models. (B) The ensemble of the 20 lowest-energy structures. Notice the lack of model convergence for the loops, suggesting that there is not a single stable lowest-energy structure.
Fig. 8.
Fig. 8.
Model structure of mdFL-PK and hFL-PK. (A) Model structure of mdFL-PK. (B) Superimposition of the model structures of mdFL-PK (red) and hFL-PK (green).
Fig. 9.
Fig. 9.
Models of medaka and human TERT–t/PK. Secondary structure of the t/PK (A, C, and E) and structural models of TERT–t/PK (B, D, and F) of Tetrahymena (A and B), medaka (C and D), and human (E and F). The structural model of Tetrahymena t/PK is based on the cryoEM structure. The color scheme for TR is same as in Fig. 1. TEN is cyan, TRBD is blue, RT is purple, and CTE is light blue. The TLFY motif within TRBD is red.
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