Analysis of the structure of human telomerase RNA in vivo - PubMed (original) (raw)
Analysis of the structure of human telomerase RNA in vivo
Mária Antal et al. Nucleic Acids Res. 2002.
Abstract
Telomerase is a ribonucleoprotein reverse transcriptase that synthesises telomeric DNA. The RNA component of telomerase acts as a template for telomere synthesis and binds the reverse transcriptase. In this study, we have performed in vivo and in vitro structural analyses of human telomerase RNA (hTR). In vivo mapping experiments showed that the 5'-terminal template domain of hTR folds into a long hairpin structure, in which the template sequence occupies a readily accessible position. Intriguingly, neither in vivo nor in vitro mapping of hTR confirmed formation of a stable 'pseudoknot' helix, suggesting that this functionally essential long range interaction is formed only temporarily. In vitro control mappings demonstrated that the 5'-terminal template domain of hTR cannot fold correctly in the absence of cellular protein factors. The 3'-terminal domain of hTR, both in vivo and in vitro, folds into the previously predicted box H/ACA snoRNA-like 'hairpin-hinge-hairpin-tail' structure. Finally, comparison of the in vivo and in vitro modification patterns of hTR revealed several regions that might be directly involved in binding of telomerase reverse transcriptase or other telomerase proteins.
Figures
Figure 1
Schematic representation of the results of in vitro (A) and in vivo (B) structure probing of the 5′-terminal domain of hTR. The structure of hTR has been adopted from Chen et al. (8) and Mitchell et al. (9). The phylogenetically conserved regions (CR1, CR2 and CR3) are boxed and the template sequence is underlined. Nucleotides modified by DMS, CMCT or kethoxal are circled in green. Positions of nuclease cleavage are indicated by arrows linked to pink squares (RNase V1, double strand-specific) and green diamonds (RNase T1, G-specific) and open (S1 nuclease, single strand-specific) and filled (RNase T2, single strand-specific) circles. The intensity of the colour indicates the intensity of chemical modification and nucleolytic cleavage. Extremely strong modifications are shown in black. Green circles with black outlines indicate altered modifications under semi-denaturing conditions. Unaffected nucleotides are not marked. Nucleotides with decreased in vivo accessibility are indicated by open (moderately protected) and filled (fully protected) blue triangles. Open and filled red stars indicate nucleotides with moderately or highly increased in vivo reactivity, respectively. Red triangles indicate unusual uridine modifications. Pauses (x) and strong stops (stop signal) of reverse transcriptase are also indicated.
Figure 2
Secondary structure analyses of the CR1 template (A), and the CR2 (B) and CR3 (C) pseudoknot regions of hTR. In vitro (lanes 1–3) and in vivo (lanes 4–6) modified hTR was analysed by primer extension using terminally labelled sequence-specific oligonucleotide primers. The extended products were fractionated on a 6% sequencing gel. Modified nucleotides are marked by red spots and listed on the right. Concentrations of chemical reagents (DMS and kethoxal) are indicated at the top. Control lanes (0) show primer extension reactions performed on non-treated RNAs. Lanes G, A, C and U represent dideoxy sequencing reactions performed with the same primer. Nucleotides with decreased in vivo accessibility are indicated by open (moderately protected) and filled (fully protected) blue triangles. Open and filled red stars indicate nucleotides with moderately or highly increased in vivo reactivity, respectively. Red triangles indicate unusual uridine modifications. Strong stops (stop signal) of reverse transcriptase are also indicated.
Figure 3
Schematic representation of the results of in vitro (A) and in vivo (B) structure probing of the box H/ACA snoRNA-like domain of hTR. The phylogenically conserved CR4, CR5, CR6, CR7 and CR8 regions are boxed. The box H and ACA sequences are underlined. Extremely strong cleavage induced by lead acetate (Pb) is marked by a black arrow. Potential and verified base pairings in the 5′ pocket are marked by red dotted and continuous lines, respectively. For other details, see the legend to Figure 1.
Figure 4
Structure probing of the box H/ACA core domain of hTR and U19 snoRNA. (A) Secondary structure of the box H/ACA core domain of hTR was analysed under in vitro (lanes 1–4) and in vivo (lanes 5–7) conditions. (B) In vivo structure probing of the box H region of human U19 snoRNA. The modification pattern of the box H region of U19 is shown at the bottom. For other details, see the legend to Figure 2.
Figure 5
Secondary structure analysis of the evolutionarily conserved CR4 (A) and CR5 (B) regions of hTR. For other details, see the legend to Figure 2.
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