Solution structure of a luteoviral P1-P2 frameshifting mRNA pseudoknot

A hairpin-type messenger RNA pseudoknot from pea enation mosaic virus RNA1 (PEMV-1) regulates the efficiency of programmed -1 ribosomal frameshifting. The solution structure and 15N relaxation rates reveal that the PEMV-1 pseudoknot is a compact-folded structure composed almost entirely of RNA triple helix. A three nucleotide reverse turn in loop 1 positions a protonated cytidine, C(10), in the correct orientation to form an A((n-1)).C(+).G-C(n) major groove base quadruple, like that found in the beet western yellows virus pseudoknot and the hepatitis delta virus ribozyme, despite distinct structural contexts. A novel loop 2-loop 1 A.U Hoogsteen base-pair stacks on the C(10)(+).G(28) base-pair of the A(12).C(10)(+).G(28)-C(13) quadruple and forms a wedge between the pseudoknot stems stabilizing a bent and over-rotated global conformation. Substitution of key nucleotides that stabilize the unique conformation of the PEMV-1 pseudoknot greatly reduces ribosomal frameshifting efficacy.     

Determinants in mammalian telomerase RNA that mediate enzyme processivity and cross-species incompatibility

Telomerase contains two essential components: an RNA molecule that templates telomeric repeat synthesis and a catalytic protein component. Human telomerase is processive, while the mouse enzyme has much lower processivity. We have identified nucleotide determinants in the telomerase RNA that are responsible for this difference in processivity. Mutations adjacent to the template region of human and mouse telomerase RNA significantly altered telomerase processivity both in vitro and in vivo. We also identified functionally important nucleotides in the pseudoknot domain of telomerase RNA that potentially mediate the incompatibility between human TERT and mouse telomerase RNA. These experiments identify essential residues of the telomerase RNA that regulate telomerase activity and processivity.     

An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of ?1 ribosomal frameshifting

An efficient −1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote −1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several −1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of −1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the −1 PRF efficiency of this stimulator can be modulated by sequential removal of base–triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem–loop interactions and their relative positions to the helical junction play crucial roles for the −1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate −1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate −1 PRF. Furthermore, it suggests that −1 PRF, induced by an in-trans RNA via specific base–triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.     

Dynamic behavior of the telomerase RNA hairpin structure and its relationship to dyskeratosis congenita

In this paper, we present the results from a comprehensive study of nanosecond-scale implicit and explicit solvent molecular dynamics simulations of the wild-type telomerase RNA hairpin. The effects of various mutations on telomerase RNA dynamics are also investigated. Overall, we found that the human telomerase hairpin is a very flexible molecule. In particular, periodically the molecule exhibits dramatic structural fluctuations represented by the opening and closing of a non-canonical base-pair region. These structural deviations correspond to significant disruptions of the direct hydrogen bonding network in the helix, widening of the major groove of the hairpin structure, and causing several U and C nucleotides to protrude into the major groove from the helix permitting them to hydrogen bond with, for example, the P3 domain of the telomerase RNA. We suggest that these structural fluctuations expose a nucleation point for pseudoknot formation. We also found that mutations in the pentaloop and non-canonical region stabilize the hairpin. Moreover, our results show that the hairpin with dyskeratosis congenita mutations is more stable and less flexible than the wild-type hairpin due to base stacking in the pentaloop. The results from our molecular dynamics simulations are in agreement with experimental observations. In addition, they suggest a possible mechanism for pseudoknot formation based on the dynamics of the hairpin structure and also may explain the mutational aspects of dyskeratosis congenita.     

Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.     

Telomerase Activity Is Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3′ Stem and Tertiary Structure

Pseudoknot formation in the core region of the telomerase RNA has been demonstrated to be important for telomerase activity in vertebrates, ciliates, and yeast. Characterization of the Saccharomyces cerevisiae telomerase RNA (TLC1) pseudoknot identified tertiary structural interactions that are also important for telomerase activity, as previously observed for the Kluyveromyces lactis and human telomerase RNA pseudoknots. In addition, the contributions of backbone ribose 2′-OH groups in the pseudoknot to telomerase catalysis were investigated previously, using 2′-OH (ribose) to 2′-H (deoxyribose) or 2′-O-methyl substitutions in the stem 2 helix, and it was proposed that one or more 2′-OH groups from the stem 2 sequences at or near the triple helix participate in telomerase catalysis. Based on these studies and investigations of the structural and thermodynamic properties of the TLC1 RNA pseudoknot region, we have examined the structural and thermodynamic perturbations of the 2′-O-methyl and 2′-H substituted pseudoknots, using UV-monitored thermal denaturation, native gel electrophoresis, and circular dichroism spectroscopy. Our results demonstrate the presence of A-form helical geometry perturbations in the backbone sugar substituted pseudoknots, show a correlation between thermodynamic stability and telomerase activity, and are consistent with the identification of the U809 ribose 2′-OH as a potential contributor to telomerase activity.     

The impact of dyskeratosis congenita mutations on the structure and dynamics of the human telomerase RNA pseudoknot domain

The pseudoknot domain is a functionally crucial part of telomerase RNA and influences the activity and stability of the ribonucleoprotein complex. Autosomal dominant dyskeratosis congenita (DKC) is an inherited disease that is linked to mutations in telomerase RNA and impairs telomerase function. In this paper, we present a computational prediction of the influence of two base DKC mutations on the structure, dynamics, and stability of the pseudoknot domain. We use molecular dynamics simulations, MM-GBSA free energy calculations, static analysis, and melting simulations analysis. Our results show that the DKC mutations stabilize the hairpin form and destabilize the pseudoknot form of telomerase RNA. Moreover, the P3 region of the predicted DKC-mutated pseudoknot structure is unstable and fails to form as a defined helical stem. We directly compare our predictions with experimental observations by calculating the enthalpy of folding and melting profiles for each structure. The enthalpy values are in very good agreement with values determined by thermal denaturation experiments. The melting simulations and simulations at elevated temperatures show the existence of an intermediate structure, which involves the formation of two UU base pairs observed in the hairpin form of the pseudoknot domain.     

The Impact of Dyskeratosis Congenita Mutations on the Structure and Dynamics of the Human Telomerase RNA Pseudoknot Domain

Abstract

The pseudoknot domain is a functionally crucial part of telomerase RNA and influences the activity and stability of the ribonucleoprotein complex. Autosomal dominant dyskeratosis congenita (DKC) is an inherited disease that is linked to mutations in telomerase RNA and impairs telomerase function. In this paper, we present a computational prediction of the influence of two base DKC mutations on the structure, dynamics, and stability of the pseudoknot domain. We use molecular dynamics simulations, MM-GBSA free energy calculations, static analysis, and melting simulations analysis. Our results show that the DKC mutations stabilize the hairpin form and destabilize the pseudoknot form of telomerase RNA. Moreover, the P3 region of the predicted DKC-mutated pseudoknot structure is unstable and fails to form as a defined helical stem. We directly compare our predictions with experimental observations by calculating the enthalpy of folding and melting profiles for each structure. The enthalpy values are in very good agreement with values determined by thermal denaturation experiments. The melting simulations and simulations at elevated temperatures show the existence of an intermediate structure, which involves the formation of two UU base pairs observed in the hairpin form of the pseudoknot domain.