A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit

Abstract

2The telomerase RNP counters the chromosome end-replication problem, completing genome 1 replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The 2 telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse 3 transcriptase (TERT). Although the majority of the 1157-nt Saccharomyces cerevisiae 4 telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, 5 containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). 6Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be active 7 in vitro and to maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not 8 support telomerase function. Using the CRISPR/dCas9-based "CARRY two-hybrid" assay to 9 assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to 10 TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo 11 complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is 12 needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-13 bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results 14 with sequence-randomized and truncated-CEH helices strongly suggest that this binding 15 interaction with TERT is dictated more by secondary than primary structure. In summary, we 16 have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the 17 catalytic core of this biomedically important RNP enzyme. 18 ABSTRACT 3 INTRODUCTION 19 20Telomeres are repetitive sequences located at the ends of linear eukaryotic chromosomes. 21While they provide critical genome-protective functions, they are unable to be fully copied by 22 DNA polymerases, owing to the end-replication problem. Short telomeres trigger a special G2/M 23 cell-cycle arrest known as senescence. In order to overcome the end-replication problem and 24 prevent senescence, most eukaryotic organisms require the ribonucleoprotein enzyme complex 25 telomerase (Greider and Blackburn, 1985). 27The telomerase core enzyme consists of a dedicated noncoding RNA subunit (TLC1 in 28 Saccharomyces cerevisiae) and a reverse transcriptase protein component (TERT, or Est2 in S. 29 cerevisiae). TERT utilizes a short template sequence in the telomerase RNA to iteratively add 30 telomere repeats to the 3¢ end of chromosomes (Greider and Blackburn, 1989). Together, these 31 two core components are sufficient to reconstitute basal telomerase activity in vitro (Beattie et 32 Zappulla et al., 2005). Telomerase RNAs are evolving strikingly fast, ranging in size 33 from ~150 nucleotides in ciliates to >2000 nucleotides in some species of yeast, with even the 34 sequences and secondary-structure models from closely related species within the same genus 35 showing limited conservation. Experiments have shown that the 1157-nt S. cerevisiae 36 telomerase RNA, TLC1, exhibits a hi...

Yeast Telomerase RNA Flexibly Scaffolds Protein Subunits: Results and Repercussions

“…This is not surprising, given that these regions make specific contacts with protein subunits. Furthermore, in the catalytic core, where TLC1 interacts with TERT [ 15 , 20 , 30 ], there are specific secondary structures that are nearly universally conserved among telomerase RNAs, including ciliates [ 47 ], from which telomerase was first isolated [ 2 ], and mammals [ 48 ], where telomerase plays a critical role in aging and cancer [ 49 , 50 , 51 ].…”

“…Examples of conserved regions in TLC1 are in the Est1 arm, where there is a Second Essential Est1-arm Domain (SEED) [ 45 ] or CS2a region [ 11 , 31 ] shown to bind to Pop1/6/7 proteins [ 28 ], and in the core, where there is an Area of Required Connectivity (ARC) [ 29 ] and core-enclosing helix (CEH) [ 30 ] ( Figure 1 ). The conserved core region of TLC1 is about the size of the ciliate telomerase RNAs (~150 nts), and this is consistent with these streamlined telomerase RNAs/RNPs (needed in vast quantities in macronuclei) being amenable to structural biology studies using cryo-EM [ 52 ].…”

“…Therefore, TLC1 RNA provides more than a template for reverse transcription and a flexible scaffold: it also has some more “conventional,” and certainly fascinating, structured elements that provide beneficial and sometimes also essential functions. In the case of the holoenzyme-specific protein subunits (e.g., Est1, Ku, Sm 7 ) of yeast telomerase RNPs, these modules have been shown to be flexibly scaffolded as reviewed in sections above, whereas, in the case of the catalytic core, there is some very interesting and impressive flexibility [ 29 , 30 ], yet it also has very clear limitations [ 29 , 30 ], consistent with the enzymatic coordination with the TERT protein and the DNA substrate.…”

Inverse-folding design of yeast telomerase RNA increases activityin vitro

“…This central core secondary structure conforms to the conserved telomerase RNA core consensus model, containing the template and templateboundary element, a pseudoknot with catalytically important base triples, and a coreenclosing helix, all coordinated through an area of required connectivity [11][12][13] . Yeast TERT, Est2 (Ever-Shorter Telomeres 2), binds to this central core and functions with it to reconstitute core-enzyme activity [13][14][15][16] .…”