UMMS Affiliation

Department of Molecular, Cell, and Cancer Biology

Publication Date

2020-11-20

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Cell Biology | Enzymes and Coenzymes | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides

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 DeltaCEH 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.

Keywords

RNA, RNP, TERT, TLC1, senescence, telomerase, telomerase RNA, telomere, two-hybrid screening, yeast

Rights and Permissions

Copyright © 2020 Mefford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

DOI of Published Version

10.1128/MCB.00239-20

Source

Mefford MA, Hass EP, Zappulla DC. A 4-Base-Pair Core-Enclosing Helix in Telomerase RNA Is Essential for Activity and for Binding to the Telomerase Reverse Transcriptase Catalytic Protein Subunit. Mol Cell Biol. 2020 Nov 20;40(24):e00239-20. doi: 10.1128/MCB.00239-20. PMID: 33046533; PMCID: PMC7685517. Link to article on publisher's site

Journal/Book/Conference Title

Molecular and cellular biology

Related Resources

Link to Article in PubMed

PubMed ID

33046533

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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