A Triple Helix within a Pseudoknot Is a Conserved and Essential Element of Telomerase RNA

Abstract

Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A ⅐ U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C ⅐ C ؉ triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C ⅐ U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerasespecific function.Telomerase, a ribonucleoprotein reverse transcriptase, makes up for losses caused by incomplete DNA replication and degradation, by adding species-specific, 5-to 26-nucleotide (nt) repeats onto the telomere termini (reviewed in reference 2). The telomerase complex contains an RNA subunit (TER) (TLC1 in Saccharomyces cerevisiae), a catalytic reverse transcriptase (TERT) (Est2 in S. cerevisiae), and several other protein components. Unlike other reverse transcriptases, telomerase specializes in repeatedly copying a short RNA template within its integral RNA component.TERs are highly divergent, being conserved in sequence only among closely related species. Phylogenetic covariation was used to predict conserved secondary structures for evolutionarily close species of ciliates (14, 24), vertebrates (4), Kluyveromyces budding yeasts of the K. marxianus cluster (31, 32), and Saccharomyces sensu stricto (3,7,13,37). Limited similarity in the general architecture was observed among these models, consisting of three long arms and a catalytic core domain (4, 13). Although nontemplate regions are essential for the assembly, regulation, and function of telomerase, their specific roles are still unclear (reviewed in reference 30). We hypothesized that important functional elements would exhibit better conservation in their tertiary structures, rather than in their secondary structures or sequences. Solving these tertiary structures may provide insights into their conserved functions.Pseudoknot elements were found to be critical for telomerase function in ciliates (28), vertebrates (4), and Kluyveromyces (32). For S. cerevisiae, alternative pseud...

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

“…The structure is phylogenetically supported (Supplemental Fig. 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.…”

A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

“…Finally and most importantly, conservation among functional domain sequences or structures provided additional confidence for the structure prediction. For example, the NMR structure of the hTR showed extensive tertiary interactions between the loop and stem nucleotides, rendering an essential triple helix in the pseudoknot structure [70,71]. A similar structure could possibly form in TbTR pseudoknot (Figure 6C).…”

Identification and comparative analysis of telomerase RNAs from Candida species reveal conservation of functional elements

“…Recently, the human, K. lactis, S. cerevisiae, and ciliate pseudoknots were proposed to contain U-AÁU base triples (Theimer et al 2005;Ulyanov et al 2005Ulyanov et al , 2007Shefer et al 2007;Qiao and Cech 2008). Given the low sequence conservation among the Candida spp.…”

“…Several conserved elements were found to contribute crucial functions such as specifying the template boundary (Tzfati et al 2000;Seto et al 2003) or recruiting Est1, the yKu complex, and the Sm proteins (Seto et al 1999(Seto et al , 2002Peterson et al 2001). A unique pseudoknot element containing a major-grove triple-helix is crucial for K. lactis, S. cerevisiae, and human telomerase function (Theimer et al 2005;Shefer et al 2007;Qiao and Cech 2008). It was recently suggested that the triple helix functions in positioning the 39 end of the telomere at the catalytic site (Qiao and Cech 2008).…”