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. 2010 Oct;38(19):6746-56.
doi: 10.1093/nar/gkq525. Epub 2010 Jun 16.

Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA

Nak-Kyoon Kim  1 , Carla A Theimer, James R Mitchell, Kathleen Collins, Juli Feigon
Affiliations

Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA

Nak-Kyoon Kim et al. Nucleic Acids Res. 2010 Oct.

Abstract

Telomerase extends the 3'-ends of linear chromosomes by adding conserved telomeric DNA repeats and is essential for cell proliferation and genomic stability. Telomerases from all organisms contain a telomerase reverse transcriptase and a telomerase RNA (TER), which together provide the minimal functional elements for catalytic activity in vitro. The RNA component of many functional ribonucleoproteins contains modified nucleotides, including conserved pseudouridines (Ψs) that can have subtle effects on structure and activity. We have identified potential Ψ modification sites in human TER. Two of the predicted Ψs are located in the loop of the essential P6.1 hairpin from the CR4-CR5 domain that is critical for telomerase catalytic activity. We investigated the effect of P6.1 pseudouridylation on its solution NMR structure, thermodynamic stability of folding and telomerase activation in vitro. The pseudouridylated P6.1 has a significantly different loop structure and increase in stability compared to the unmodified P6.1. The extent of loop nucleotide interaction with adjacent residues more closely parallels the extent of loop nucleotide evolutionary sequence conservation in the Ψ-modified P6.1 structure. Pseudouridine-modification of P6.1 slightly attenuates telomerase activity but slightly increases processivity in vitro. Our results suggest that Ψs could have a subtle influence on human telomerase activity via impact on TER-TERT or TER-TER interactions.

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Figures

Figure 1.
Figure 1.
Secondary structure of hTER and structure of Ψ. (A) Comparison of the structures of uridine and Ψ. (B) Secondary structure of the unmodified P6.1 (left) and Ψ modified (right) domains. Right: the naturally occurring Ψ modification sites are shown in red and two Ψs in the stem substituted during in vitro RNA synthesis in bold black. Left: upper case green, lower case green and black residues stand for 100%, ≥80% and <80% conserved nucleotides in vertebrate TER, respectively. In both P6.1 and Ψ4-P6.1, the terminal G-C pairs (box) are not from the natural hTER sequence. Secondary structures of hTER showing the pseudoknot/core, CR4-CR5, CR7 and H/ACA domains. Putative Ψ modification sites are indicated by arrows, with the P6.1 sub-domain circled in red.
Figure 2.
Figure 2.
Identification of Ψ modification sites in hTER. Ψs were detected as stops to primer extension on modified RNA from a HeLa extract fraction enriched for endogenous hTER. Lane 1: untreated RNA as template; lanes 2 and 3: CMC mock-treated or CMC-treated RNA as template. The stops to RT that occur one nucleotide before potential sites of Ψ modification are marked with asterisks. Lanes 4–7: RT of recombinant template was performed with ddNTP mixes to generate a sequencing ladder. The non-specific background pattern is an artifact of scanning a long radiographic exposure at high gain, which was required to visualize the weak signals. The RT product of complete synthesis to the hTER 5′-end is indicated (FL-ehTER). (A) Primer extension using primer complementary to hTER nucleotides 443–419. (B) Primer extension using primer complementary to hTER nucleotides 235–211.
Figure 3.
Figure 3.
NMR spectra of Ψ4-P6.1 and comparison with P6.1. (A) 1D imino (600 MHz, 10°C) and 2D NOESY (600 MHz, 10°C, τm = 250 ms) spectra of Ψ4-P6.1. (B) Comparison of NOESY spectra of Ψ4-P6.1 (black, 600 MHz, τm = 200 ms) and P6.1 (red, 800 MHz, τm = 150 ms) at 20°C. Large chemical shift differences are observed for the residues 305, 308, 309 and 310 as well as between Ψs and Us.
Figure 4.
Figure 4.
NMR solution structure of pseudouridylated P6.1 (Ψ4-P6.1). (A) Superposition of the 20 lowest energy structures over all heavy atoms. (B) Stereo view of the lowest energy structure of Ψ4-P6.1. Phosphate backbone is outlined by a gray ribbon. In both (A) and (B), nucleotides A, Ψ, G and C are colored by yellow, green, blue and red, respectively.
Figure 5.
Figure 5.
Detailed tertiary structure of the loop regions of Ψ4-P6.1 and P6.1. Stereo view of the loop from the lowest energy structure of (A) Ψ4-P6.1 and (B) P6.1 [PDB ID: 1OQ0; (32)]. Nucleotides are color-coded as shown in Figure 2B. Comparison of U•G wobble pairs in (C) Ψ4-P6.1, (D) P6.1 and (E) the UUCG tetraloop [PDB ID: 1F7Y; (56)].
Figure 6.
Figure 6.
Direct telomerase activity assays of in vitro reconstituted telomerase with hTERT and hTER components. (A) Direct assays with FL-hTER (nucleotides 1–451), pseudoknot/core + CR4-CR5, pseudoknot/core + CR5318 and core + Ψ3CR5318 are shown from the lanes 2–5. Lane 1: mock reaction without RNA. The 15-nt 32P-5′-end labeled DNA loading control (L.C.) is indicated. (B) The secondary structure of CR5318 with sites of Ψ modification marked with wedge symbols. Relative telomerase activities (C) and processivities (D) are shown for the RNAs listed in (A). The processivity = −ln2/(2.303k), where k is the slope of a linear fit (61).
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