Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

Rachel O Niederer, David C Zappulla, Rachel O Niederer, David C Zappulla

Abstract

Telomerase catalyzes the addition of nucleotides to the ends of chromosomes to complete genomic DNA replication in eukaryotes and is implicated in multiple diseases, including most cancers. The core enzyme is composed of a reverse transcriptase and an RNA subunit, which provides the template for DNA synthesis. Despite extensive divergence at the sequence level, telomerase RNAs share several structural features within the catalytic core, suggesting a conserved enzyme mechanism. We have investigated the structure of the core of the human and yeast telomerase RNAs using SHAPE, which interrogates flexibility of each nucleotide. We present improved secondary-structure models, refined by addition of five base triples within the yeast pseudoknot and an alternate pairing within the human-specific element J2a.1 in the human pseudoknot, both of which have implications for thermodynamic stability. We also identified a potentially structured CCC region within the template that may facilitate substrate binding and enzyme mechanism. Overall, the SHAPE findings reveal multiple similarities between the Saccharomyces cerevisiae and Homo sapiens telomerase RNA cores.

Keywords: RNA; RNP; SHAPE; TLC1; chemical probing; hTR; lncRNA; ncRNA; telomerase; telomerase RNA.

© 2015 Niederer and Zappulla; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Figures

FIGURE 1.
FIGURE 1.
SHAPE-directed secondary-structure model of the core of S. cerevisiae telomerase RNA. (A) The refined yeast telomerase RNA secondary-structure model incorporating SHAPE results. GU wobble pairs indicated by open circles. Other non-Watson–Crick interactions are denoted with filled circles. Open triangles correspond to deletions relative to TLC1 and exogenous sequence is lowercase. (B) SHAPE reactivity at each position in the yeast telomerase RNA core. Values represent the average of between 5 and 18 independent trials with standard error shown.
FIGURE 2.
FIGURE 2.
Pseudoknot-disrupting mutants increase reactivity in stem 1. (A) Design of pseudoknot-disrupting mutants. Seven nucleotides in stem 1 were mutated to their Watson–Crick cognate to abolish base-pairing. The mutations are predicted to disrupt pseudoknot formation without affecting the alternately observed two stem–loop conformation (Liu et al. 2012) (right). Pseudoknot-disrupting mutants are indicated as dmPK-1 and dmPK-2. (B) SHAPE reactivity in WT and PK-disrupting mutants. SHAPE reactivity is plotted for WT (from Fig. 1), both PK-disrupting mutants and the stem 1 restoring mutant dmPK-R. Reactivities for dmPK-1, dmPK-2, and dmPK-R correspond to the average value between 3 and 9 independent trials with standard deviation shown. Mutated nucleotides are indicated by brackets. (C) Pseudoknot-disrupting mutants decrease telomerase activity in vitro. Telomerase assay testing activity of PK and compensatory mutants. Alignment of DNA primer with TLC1 is shown. (TBE) template-boundary element.
FIGURE 3.
FIGURE 3.
SHAPE-refined secondary-structure model of the H. sapiens core. (A) SHAPE reactivity mapped onto hTR secondary structure. Non-Watson–Crick interactions indicated by circles with GU wobble pairs denoted by open circles. Hoogsteen-face interaction is denoted with square. Dashed lines indicate reactive residues previously shown to pair in vitro (Zhang et al. 2011). (B) SHAPE reactivity by nucleotide. Values represent the average of between 6 and 12 independent trials. Standard error is shown.
FIGURE 4.
FIGURE 4.
Models of the yeast and human TR cores. (A) Micro-TLC1 and the hTR core show similar reactivity patterns. Summary diagram of general reactivity patterns for each core. Regions of low reactivity common to both hTR and Micro-TLC1 are shown in black, while common regions of high reactivity are colored red. (B) A general model for telomerase RNA core domain coordination with TERT. A generic telomerase RNA core with common structural elements is shown. Regions that may introduce physical flexibility based on SHAPE data are highlighted in red. The potential conserved structural element consisting of CCC nucleotides in the template is drawn in proximity to the catalytically important base triples and ending in the active site, shown as an open oval. The template is indicated by a thick line. Telomeric DNA is shown in blue.

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