Mutations linked to dyskeratosis congenita cause changes in the structural equilibrium in telomerase RNA

Abstract

Autosomal dominant dyskeratosis congenita (DKC), as well as aplastic anemia, has been linked to mutations in the RNA component of telomerase, the ribonucleoprotein responsible for telomere maintenance. Here we examine the effect of the DKC mutations on the structure and stability of human telomerase RNA pseudoknot and CR7 domains by using NMR and thermal melting. The CR7 domain point mutation decreases stability and alters a conserved secondary structure thought to be involved in human telomerase RNA accumulation in vivo. We find that pseudoknot constructs containing the conserved elements of the pseudoknot domain are in equilibrium with a hairpin conformation. The solution structure of the wild-type hairpin reveals that it forms a continuous helix containing a novel run of three consecutive U⅐U and a U⅐C base pairs closed by a pentaloop. The six base pairs unique to the hairpin conformation are phylogenetically conserved in mammals, suggesting that this conformation is also functionally important. The DKC mutation in the pseudoknot domain results in a shift in the equilibrium toward the hairpin form, primarily due to destabilization of the pseudoknot. Our results provide insight into the effect of these mutations on telomerase structure and suggest that the catalytic cycle of telomerase involves a delicate interplay between RNA conformational states, alteration of which leads to the disease state. D yskeratosis congenita (DKC) is a rare inherited multisystemic disorder characterized by abnormal skin pigmentation, leukoplakia, and nail dystrophy (1). The known phenotypic hallmarks of DKC are defects in highly proliferative tissues that could ensue from a telomere maintenance disorder (2, 3). The leading causes of premature mortality due to DKC are progressive bone marrow failure, pulmonary disease, and malignancy. X-linked, autosomal recessive, and autosomal dominant inheritance patterns have been observed for DKC, and telomere lengths in patients with DKC are found to be significantly reduced in all three forms of inheritance (4). Similarly, some patients with ideopathic aplastic anemia also have shorter telomeres than age-matched controls (5). Although the X-linked form of DKC is characterized by mutations in the gene encoding the protein dyskerin (6), a component of the telomerase ribonucleoprotein (7), the autosomal dominant form of DKC has been linked to mutations in the gene encoding human telomerase RNA (hTR). The presence of telomerase RNA mutations and their segregation with the disease were confirmed in three families with DKC inheritance (8). One mutation results in deletion of the H͞ACA and CR7 domains (9) required for nucleolar localization, 3Ј-end processing, and RNA stability (7,(10)(11)(12), whereas the second is a point mutation (C408G) in the CR7 domain (Fig. 1a). The third mutation is a two-base substitution in the essential pseudoknot domain (9), which is required for activity (13,14) and is involved in the binding of the protein catalytic subunit (10) (Fig. 1a). Some patients with aplastic ...

Functional analysis of the pseudoknot structure in human telomerase RNA

“…1B). In this model, the RNA structure switches between the P3 pseudoknot and the intraloop base pairing conformations during telomerase elongation (7,8). However, in our minimal RNA reconstitution system, telomerase activity was fully restored by using a short 14-mer RNA oligonucleotide (nucleotides 171-184) that forms the P3 helix in trans with the P2 stem-loop RNA fragment (nucleo- tides 44 -147) (Fig.…”

“…An NMR solution structure of a short RNA oligonucleotide representing the P2b stem-loop (nucleotides 93-122) indicated that the loop region of this RNA can form four noncanonical pyrimidine:pyrimidine base pairings and two Watson-Crick base pairings when the complementary strand of the P3 helix is not present (7). The intraloop base pairing was thus suggested to be in thermodynamic equilibrium with the P3 helix (Fig.…”

“…A ''molecular switch'' hypothesis for pseudoknot function was proposed based on a NMR structure of the P2b stem-loop (Fig. 1B) (7,8). This model predicted a conformational conversion between the pseudoknot inferred from the phylogenetic study and the intraloop base pairings seen in the NMR structure of the P2b-stem-loop RNA (nucleotides 93-122).…”

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

“…The wild-type hTR, which contains the U177 single-nucleotide bulge (Fig. 1g), is in dynamic equilibration with a hairpin conformation found between nts 93-121 (36). Deletion of U177 stabilizes the pseudoknot, thus eliminating the switch to the alternate conformation (28).…”

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

“…However, direct structural proof for the pseudoknot fold in the full-length telomerase RNA and RNP has not yet been demonstrated. Moreover, the conformation adopted by the sequence within the active telomerase enzyme and its behavior during catalysis remain incompletely understood, though it has been hypothesized to act as a "molecular switch" in promoting enzyme translocation during processive repeat addition (17,29,32,33).In this work, we used single-molecule FRET (34-36) to interrogate the conformation adopted by the Tetrahymena pseudoknot sequence and monitored its behavior during catalysis. To determine whether the sequence can fold into a pseudoknot, we excised the region from the rest of the telomerase RNA and characterized its conformation in isolation, demonstrating that the sequence can indeed fold into a stable pseudoknot.…”