Abstract
The telomerase ribonucleoprotein (RNP) counters the chromosome end replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (telomerase reverse transcriptase [TERT]). Although the majority of the 1,157-nucleotide (nt) Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4 bp of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas the ΔCEH and 1- and 2-bp alleles do not support telomerase function. Using the CRISPR/nuclease-deactivated Cas9 (dCas9)-based CARRY (CRISPR-assisted RNA-RNA-binding protein [RBP] yeast) two-hybrid assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nt that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices suggest that this binding interaction with TERT is dictated more by secondary than by primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.
Highlights
Telomeres are repetitive sequences located at the ends of linear eukaryotic chromosomes.While they provide critical genome-protective functions, they are unable to be fully copied by DNA polymerases, owing to the end-replication problem
Our previous results suggested that the core-enclosing helix is required for activity in vitro in the context of a circularly permuted telomerase RNA allele comprising just the catalytic core, Micro
To further test the conclusion that the core-enclosing helix is essential in yeast telomerase, we examined telomerase function when the CEH was deleted in a different circular permutant, cpTBE
Summary
Telomeres are repetitive sequences located at the ends of linear eukaryotic chromosomes.While they provide critical genome-protective functions, they are unable to be fully copied by DNA polymerases, owing to the end-replication problem. TERT utilizes a short template sequence in the telomerase RNA to iteratively add telomere repeats to the 3¢ end of chromosomes (Greider and Blackburn, 1989). Together, these two core components are sufficient to reconstitute basal telomerase activity in vitro (Beattie et al, 1998; Zappulla et al, 2005). TLC1 acts as a flexible scaffold to bind the holoenzyme proteins in the RNP enzyme complex: i.e., the binding sites for Est[1], Ku, and Sm7 can each be repositioned to novel locations within the RNA while supporting these subunits’ functions (Zappulla and Cech, 2004; Zappulla et al, 2011; Mefford et al, 2013; Hass and Zappulla, 2017). Large portions of the RNA are dispensable for function in vivo and in vitro (Livengood et al, 2002; Zappulla et al, 2005; Qiao and Cech, 2008; Mefford et al, 2013)
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