A molecular switch underlies a human telomerase disease - PubMed (original) (raw)

A molecular switch underlies a human telomerase disease

Luis R Comolli et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Telomerase is a ribonucleoprotein (RNP) required for maintenance of telomeres. Although up-regulated telomerase activity is closely linked to the cellular immortality characteristic of late stage carcinogenesis, recently, mutations in the telomerase RNA gene in humans have been associated with dyskeratosis congenita and aplastic anemia, both typified by impaired haemopoietic function. These mutations include base changes in a highly conserved putative telomerase RNA pseudoknot. Here, by using in vitro telomerase assays, NMR, and UV absorbance melting analyses of model oligonucleotides designed to form a "trans-pseudoknot," we describe functional, structural, and energetic properties of this structure. We demonstrate that the pseudoknot domain exists in two alternative states of nearly equal stability in solution: one is the previously proposed pseudoknot formed by pairing P3 with the loop domain of P2b, and the other is a structured P2b loop alone. We show that the two-base mutation (GC1078 --> AG) present in one gene copy in a family with dyskeratosis congenita abrogates telomerase activity. This mutation hyperstabilizes the P2b intraloop structure, blocking pseudoknot formation. Conversely, when the P3 pseudoknot pairing is hyperstabilized by deleting a conserved bulge in P3, telomerase activity also decreases. We propose that the P2bP3 pseudoknot domain acts as a molecular switch, and interconversion between its two states is important for telomerase function. Phylogenetic covariation in the P2b and P3 sequences of 35 species provides a compelling set of "natural" compensatory base pairing changes supporting the existence of the crucial molecular switch.

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Figures

Fig 1.

Proposed vertebrate telomerase RNA secondary structure and model oligonucleotide systems studied. (a) Schematic diagram of putative core secondary structure (8). CR2 to CR8 are conserved regions. The pseudoknot domain is formed between CR2, the long stem-loop that includes P2b, and CR3, which includes P3. (b) Representative telomere repeat amplification protocol assay of telomerase reconstituted in vivo in VA13 human cells, from indicated hTR mutants. “H” denotes that telomerase was inactivated by heat (80°C, 10 min), dys + dys* contains both RNA species, and dys/dys* contains RNA with both dyskeratosis and P3 compensatory mutations in the same molecule. (c) P2b sequence (black). Residues 99–115 comprise loop J2b/3 in hTR. Putative pseudoknot formed with P2b and P3 (blue) and a possible pairing with CR5 (green). Residue numbers are from full-length hTR. Two mutations in one family with dyskeratosis, G107A and C108G, are shown by red and arrows. P3-mutant deletes the putative U177 bulge in P3. (d) Small oligonucleotides comprising the P2b moiety proposed to base pair with P3, 1/2P2b, and control 2/3P2b with CR5.

Fig 2.

Complex between P2b and P3 oligonucleotides in nondenaturing polyacrylamide gels. Lane 1, free P2b. Lane 2, P2b and P3 (1:1), with slight P3 excess providing a marker. Lane 3, P2b and P3-mutant (1:1). Lane 4, P3 and P3-mutant competing for binding to P2b (1:1); wild-type P3 is outcompeted. Lane 5, P2b and CR5 (1:1). Lane 6 shows association of P2b and P3, and lane 7 shows association of P2b and P3-mutant in the presence of CR5. Lane 8, 1/2P2b with P3. Lane 9, 1/2P2b with P3-mutant. Lane 10, P3, P3-mutant, and CR5 (1:1:1). P3-mutant runs slightly faster than P3; both bands overlap at high RNA concentrations. Lane 11, free 1/2P2b.

Fig 3.

Comparison of the relative stabilities of P2b, P2b-dys, and the “_trans_-pseudoknot.” (a) Derivative of UV melting curves for P2b (blue; _T_m = 50.6°C, 78.8°C), P2b:P3 (magenta; _T_m = 49.9°C, 78.5°C), P2b:P3-mutant (red; _T_m = 51.0°C, 78.5°C), and P2b:CR5 (green; _T_m = 50.3°C, 79.5°C) in equimolar ratios, each oligonucleotide at 2.4 μM. (b) Same as in a but RNA at 275 μM. P2b (_T_m = 48.7°C, 77.7°C), P2b:P3 (_T_m = 46.1°C, 75.5°C), P2b:P3-mutant (_T_m = 58.6°C, 79.2°C), and P2b:CR5 (_T_m = 46.0°C, 73.4°C). (c) Short double-stranded RNA constructs 1/2P2b:P3 (dashed magenta; _T_m = 38.7°C) and 1/2P2b:P3-mutant (dashed red; _T_m = 46.0°C). (d) Comparison of melting curve derivatives for P2b (blue, _T_m = 50.6°C, 78.8°C) and P2b-dys (dashed blue, _T_m = 54.2°C, 77.0°C). After dialysis against NMR buffer, 1 mM MgCl2 was added in each case, resulting in a lower Mg-to-RNA ratio for the high RNA concentration samples. Notice in a and b a lower _T_m at a high concentration for each P2b domain.

Fig 4.

Association of P2b and P3 monitored by NMR. Imino proton region of P2b, P2b:P3, and P2b:CR45.

Fig 5.

Formation of a pseudoknot in trans monitored by NOESY. Imino proton region for P2b (a) and P2b:P3 (b) in 1:1 ratio, showing disappearance of internal loop cross-peaks when P2b associates with P3 and appearance of a new set of cross-peaks. (c) Dyskeratosis mutant of P2b. The three UU base pairs of the loop give cross-peaks identical to P2b. Other cross-peaks disappear, indicated by blue dashed lines.

Fig 6.

Species covariation of residues forming Watson–Crick base pairs in the human sequence of the molecular switch. (a) Watson–Crick base pairs in both P3 (Upper) and P2b intraloop (Lower) interactions. (b) Lower stability in the P3-P2b interaction is more than compensated by lower stability in the P2b intraloop. (c) P3 has a bulged C, rather than a bulged U, and consequently lower stability to balance the loss of Watson–Crick base pairing in the P2b intraloop. (d) P3 has a bulged A, rather than a bulged U, plus an extra bulge with consequently lower stability to balance the loss of Watson–Crick base pairing for the P2b intraloop.

Fig 7.

A human telomerase RNA molecular switch mediated by the pseudoknot domain. In this model, two states are equally important: (a) in the open state of wild-type human telomerase RNA, the J2b/3 loop is free but internally structured; (b) in the alternative closed state, loop residues base pair with P3. The stability of the P3 interaction is comparable to the stability of the internal structure of loop J2b/3, so both states can interconvert. The dyskeratosis mutation G107A, C108G increases loop stability while simultaneously decreasing the potential number of P3 base pairs, so the pseudoknot does not form. Deletion of U177 hyperstabilizes the pseudoknot. Both mutations impair telomerase activity.

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A molecular switch underlies a human telomerase disease - PubMed (original) (raw)