Structure and sequence elements of the CR4/5 domain of medaka telomerase RNA important for telomerase function

Abstract

Telomerase is a unique reverse transcriptase that maintains the 3' ends of eukaryotic chromosomes by adding tandem telomeric repeats. The RNA subunit (TR) of vertebrate telomerase provides a template for reverse transcription, contained within the conserved template/pseudoknot domain, and a conserved regions 4 and 5 (CR4/5) domain, all essential for catalytic activity. We report the nuclear magnetic resonance (NMR) solution structure of the full-length CR4/5 domain from the teleost fish medaka (Oryzias latipes). Three helices emanate from a structured internal loop, forming a Y-shaped structure, where helix P6 stacks on P5 and helix P6.1 points away from P6. The relative orientations of the three helices are Mg2+ dependent and dynamic. Although the three-way junction is structured and has unexpected base pairs, telomerase activity assays with nucleotide substitutions and deletions in CR4/5 indicate that none of these are essential for activity. The results suggest that the junction is likely to change conformation in complex with telomerase reverse transcriptase and that it provides a flexible scaffold that allows P6 and P6.1 to correctly fold and interact with telomerase reverse transcriptase.

Figure 1.

Secondary structure of medaka TR. Schematic of secondary structure of medaka TR with CR4/5 domain colored in blue. mdCR4/5 sequence used in the NMR study and base pairs predicted by phylogenetic comparative analysis are shown in the box. The 100% conserved nucleotides in the five identified teleost fish TR are highlighted in red (30).

Figure 2.

Identification of stem and junction base pairs in mdCR4/5. (A) Imino proton region of 2D ¹H-¹H NOESY spectrum reveals the base pairs of mdCR4/5. NOE cross-peaks between the neighboring imino protons are indicated for P5 (cyan), P6 (green), P6.1 (orange) and the junction (magenta). Three distinct canonical G•U wobble pairs are identified; U195•G180 is in the P6 helix, and G175•U215 and G198•U212 are in the junction. The 2D HNN COSY spectra in (B) show a direct correlation between imino protons and their hydrogen-bonded nitrogen atoms in the canonical base pairs (upper box: G-C, lower box: A-U). Cross-peak labels are colored as in (A).

Figure 3.

NMR solution structure of mdCR4/5. (A) Superposition of the 20 lowest energy structures. (B) Stereo view of the lowest energy structure. The junction (J) is classified as J_5/6, J_6/6.1 and J_6.1/5 based on their flanking subdomain names. U182 is highlighted in red. (C) Schematic of the tertiary fold of mdCR4/5. The phosphate backbone is indicated by gray line, base pairs are shown as bars and the unpaired nucleotides are shown as half bars. P6, P6.1, P5 and J are colored as in Figures 1 and 2.

Figure 4.

Structure of the three-way junction and P6.1 loop of mdCR4/5. (A) Stereo view of the junction in the lowest energy structure. Hydrogen bonds in base pairs are shown by dotted lines. (B) Schematic showing the base interactions in the three-way junction. (C) Structure of the P6.1 loop. Nucleotides A, U, G and C are colored yellow, green, blue and red, respectively.

Figure 5.

Mg²⁺ ion induces a change in the relative orientations of the P5, P6 and P6.1 helices (A) ¹H-¹³C HSQC spectra (C8H8 and C6H6 regions) of mdCR4/5 as a function of added Mg²⁺. MgCl₂ was added to a final concentration (mM) of 0 (red), 5 (green), 10 (yellow) and 20 (blue) with 0.5 mM mdCR4/5. (Inset) Sites of divalent cation localization as determined from paramagnetic line broadening in the presence of Mn²⁺ are colored in blue (see also Supplementary Figure S2) (B) Superposition of the 20 lowest energy structures of mdCR4/5 structure refined using RDCs measured for the sample with Mg²⁺ (mdCR4/5-Mg²⁺). (C) Stereo-view of the lowest energy structure of mdCR4/5-Mg²⁺. For B and C, subdomains are colored as in Figure 2. (D) Lowest energy structure of mdCR4/5-Mg²⁺ with regions of divalent cation localization shown in blue. (E) Superposition of lowest energy structures of mdCR4/5 (red) and mdCR4/5-Mg²⁺ (gray). Superposition is on P6 residues 178–197, and arrows indicate changes in the positions of P6.1 and P5.

Figure 6.

Telomerase activity assays of mdCR4/5 mutants. (A) Secondary structure of mdCR4/5 with junction nucleotides that were changed highlighted in bold, base pairs that were changed shown boxed, substitutions in the P6.1 loop indicated by arrows and the U182C bulge mutation in P6 indicated by a wedge. (B) Effect of mdCR4/5 mutations on telomerase activity. Except for lane 1, full-length mdTERTs synthesized in RRL were assembled with full-length mdTR mutants. Relative activities compared with wild-type (WT) mdTR are shown in bold below the lane numbers and errors in the parentheses. (C) Electrophoretic mobility shift assay of the mdCR4/5 domain mutants. ‘Repair’ in (B) and (C) stands for simultaneous substitutions of G175U-C176U-G198U and U212A-G213A-U215A.