Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo

Abstract

Telomerase is a ribonucleoprotein complex that extends the 3′ ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C + •G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.RNA triplex | yeast | RNA structure | Hoogsteen T elomerase is a ribonucleoprotein complex that extends the 3′ ends of eukaryotic chromosomes by adding successive telomere DNA repeats using an internal RNA template and a specialized reverse transcriptase (1, 2). Telomeres are the protein-DNA complexes that form the ends of linear chromosomes and protect them from end-to-end fusion and degradation (3, 4). Telomerase is of significant medical interest owing to the correlation between telomere length and human health and the association of telomerase activity with cancer (5, 6). In the absence of telomerase activity, telomeres shorten with each round of cell division because of exonuclease digestion and the inability of conventional DNA polymerases to fully replicate linear chromosomes. Shortening past a critical length leads to cell cycle arrest and/or apoptosis (7). Telomerase activity is undetectable in most somatic cells, resulting in telomere attrition with each cell cycle (8, 9). On the other hand, telomerase is active in, and essential for the proliferation of, the germ line, some epithelial, haemopoietic, and stem cells, as well as ∼90% of cancer cell lines (10, 11). A number of inherited diseases are associated with telomere shortening due to telomerase insufficiency, such as dyskeratosis congenita, aplastic anemia, and pulmonary fibrosis (12-15).The telomerase holoenzyme consists of the telomerase reverse transcriptase (TERT) and telomerase RNA (TER), which are essential and sufficient for catalytic activity in vitro (16), and several species-specific accessory proteins. TERs are highly divergent in size and sequence between species, ranging from ∼150 nt in ciliates, ∼450 nt in vertebrates, to more than 2,000 nt in some fungi (17). TERs provide the template for telomeric DNA synthesis but also contain other domains that are essential for telomerase ass...

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

“…1), however, mutations in this region in the context of larger yeast telomerase RNAs have resulted in only a slight decrease in activity in vivo and in vitro (Lin et al 2004;Shefer et al 2007;Qiao and Cech 2008). These base triples have been previously proposed based on thermodynamic characterization and were observed in Kluyveromyces lactis (Liu et al 2012;Cash et al 2013) and their main function is likely to favor pseudoknot formation and stability without directly contributing to catalysis.…”

Structural determinants for ligand capture by a class II preQ 1 riboswitch

“…3C), where the loop 1-stem 2 base pairs stack on stem 1. Triple helices formed between loop 1 and stem 2 at the junction between the two stems of an H-type pseudoknot are a frequent feature in, for example, telomerase pseudoknots (41,42) and other riboswitches (33-35, 43, 44).…”

“…In the NMR conditions (60 mM KCl, 3 mM CaCl 2 ; pH 6.3), the riboswitch construct used for NMR (WT) has a K D of 75 ± 10 nM, and the full-length riboswitch including P1 (WT+P1) has a slightly weaker K D of 220 ± 30 nM. Deletion of residues 36-49 [ΔP4 (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)], which include all of P4 and the two flanking C residues, from the NMR construct decreased the binding affinity by 20-fold, to 2,000 ± 300 nM. A similar result (K D = 2,000 nM) was obtained from 2-aminopurine fluorescence measurements on the Spn preQ 1 -II riboswitch with only P4 deleted (equivalent residues 37-48) (40).…”

“…Examination of NMR spectra of ΔP4 (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) showed that deletion of P4 stabilizes P3 base pairs; imino proton resonances for the top five of the six base pairs of P3 were seen in the absence of preQ 1 (Fig. S4B), whereas in the WT construct only A55-U14, G54-U15, and G53-C16 base pairs were observed (Fig.…”

“…Two hundred initial structures of preQ 1 riboswitch bound to preQ 1 were calculated using Xplor-NIH 2.9.8 starting from an extended, unfolded RNA oligomer using NOE distance and dihedral angle restraints, quantified as described previously (33,41,42), following standard Xplor protocols. The 100 lowest-energy structures were further refined.…”