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

The telomerase 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 (TERT). Although the majority of the 1157-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-base pairs of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based “CARRY 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 nucleotides 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 strongly suggest that this binding interaction with TERT is dictated more by secondary than 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.


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Telomeres are repetitive sequences located at the ends of linear eukaryotic chromosomes.

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While 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).

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The 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

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In contrast to other conserved core elements, very little is known about the core-enclosing 51 helix's function. Being centrally located within the ARC, the CEH physically connects the 52 pseudoknot to the template, enclosing the telomerase RNA's core. However, when the CEH is 53 deleted in the context of a functional circular permutation (i.e., maintaining RNA backbone 54 integrity through the ARC), telomerase is inactivated in vitro (Mefford et al., 2013), suggesting a 55 key role for the core-enclosing helix beyond simply enclosing the core. Consistent with the CEH 56 being essential, large deletions that encompass either the 5¢ or 3¢ side of the core-enclosing 57 helix cause senescence and disrupt Est2 binding in vivo (Livengood et al., 2002). Also 58 suggesting that the CEH is a TERT-binding region, the CEH region has been shown to interact 59 physically with a protein via gPAR-CLIP (Freeberg et al. 2013).

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Here, we set out to investigate the structural and functional requirements of the core-enclosing 62 helix in S. cerevisiae. We find that a core-enclosing helix of 4 base pairs is sufficient to provide 63 telomerase function in yeast by promoting binding to TERT. There appear to be no sequence-64 specific requirements within the helix, indicating that TERT is generally binding double-stranded 65 DNA. Together, these data convey the importance of the core-enclosing helix in yeast, while 66 simultaneously explaining its sequence changes during evolution.

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The core-enclosing helix is required to prevent senescence and support telomerase 73 activity 74 75 Our previous results suggested that the core-enclosing helix is required for activity in vitro in the 76 context of a circularly permuted telomerase RNA allele comprising just the catalytic core, Micro-77 T(170) (Mefford, Rafiq, and Zappulla 2013) (Fig. 1A). To investigate whether the core-enclosing 78 helix is essential in vivo and to elucidate the structural requirements for function, we deleted or 79 truncated the core-enclosing helix (CEH) in a circularly permuted larger telomerase RNA allele

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We observed that the cpJ3 allele supported growth through 275 generations, as shown 89 previously (Mefford et al., 2013), whereas the cpJ3DCEH allele (Fig. 1C) senesced by 75 90 generations, similarly to a Dtlc1 strain ( Fig. 2A). Senescing cpJ3DCEH cells exhibited telomeres 91 that were shorter than the cpJ3 cells on telomere Southern blots ( Fig. 2B; lanes 13 and 14). The 92 inability of cpJ3∆CEH cells to maintain telomeres did not seem to be due to RNA abundance, 93 since we detected nearly the same level of DCEH RNA by northern blotting as the functional 94 cpJ3 allele (Fig. 2C). Furthermore, we observed that reconstituted telomerase (Zappulla et al., 6 2005) using cpJ3DCEH was catalytically dead in vitro (Fig. 2D). Thus, the core-enclosing helix is 96 required for telomerase activity and function in cells.

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To further test the conclusion that the core-enclosing helix is essential in yeast telomerase, we 99 examined telomerase function when the CEH was deleted in a different circular permutant,    Given the functional necessity of the core-enclosing helix, we set out to determine what features 113 of its structure are required. We began by adding back the native core-proximal base pairs one 114 by one (Fig. 1C). This analysis revealed that just 4-base pairs of native core-enclosing helix  those that did survive) (Fig. 3A), and without clearly perceptible telomerase activity (Fig. 3D,   119 lane 6). In contrast, 1-and 2-bp core-enclosing helices in cpJ3 did not support any telomerase 7 function in vitro or in vivo (Fig. 2). For these alleles, it is likely that base pairing does not stably 121 form. Thus, our data show that a minimum of 4 base pairs is required for substantial core-122 enclosing helix function.

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Adding back more of the native core-enclosing helix also supported telomerase activity in vivo  To determine whether the sequence of the native core-enclosing helix sequence is important, 137 we tested non-native base pairs. Interestingly, a sequence-randomized 4-bp helix ( Fig. 1C; 138 GAAU vs. wild-type CUGA) was able to support weak telomerase activity in the context of 139 cpTBE, both in vivo ( Fig. 3A; Fig. 3B, lanes 21-23) and in vitro (Fig. 3D, lane 10). The same 140 random-sequence 4-bp helix in the context of cpJ3 was not sufficiently functional in vivo to 141 prevent senescence ( Fig. 2A), although it had weakly perceptible activity in vitro (Fig. 2D). A 142 longer 16-bp random helix (Fig. 1C) in cpTBE showed similar telomerase function to the 4-bp 143 random helix (Fig. 3D). These results suggested that the endogenous sequence of the core-144 enclosing helix has a nonessential role in its function.

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The core-enclosing helix is required for telomerase RNA binding to TERT

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What is the mechanistic role of the core-enclosing helix in telomerase RNP function? Our 176 results show that the CEH is required for both in vivo and in vitro telomerase action -thus, the 177 CEH is essential for fundamental telomerase core-enzyme activity. Since the core enzyme is 178 composed of the TLC1 RNA and the TERT catalytic protein subunit, a parsimonious explanation 179 for the contribution of CEH to enzyme function is that it is simply required for the RNA to bind to 180 TERT to assemble the core enzyme.

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To test the hypothesis that the CEH is a key binding site for TERT in telomerase RNA, we used   Micro-T cpTBE with a 1-bp, 2-bp, or 3-bp CEH did not bind TERT, but a 4-bp CEH did, 10 consistent with performance of these alleles in telomerase function in vivo and in vitro (Figs. 2 197 and 3). We also tested binding by the random 4-bp and 23-nt CEH alleles and these showed 198 little or no HIS activation. Presumably these alleles bind intermediately to TERT; too weakly for 199 the CARRY two-hybrid system to detect (³ ~1 µM Kd; ), yet still 200 sufficient to provide telomerase assembly and function, as shown in Figure 2 and 3. Overall, we 201 conclude that the CEH is required to bind to TERT and that 4 base pairs is sufficient for this 202 critical interaction at the core of the telomerase RNP.

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In order to study the core-enclosing helix, which is in the middle of the Area of Required 213 Connectivity, we moved the ends of telomerase RNA away from the ARC so as not to disrupt it 214 in our CEH-mutant alleles. Thus, we circularly permuted TLC1 RNA, allowing us to delete the 215 CEH without disrupting RNA continuity through the ARC. The ability to parse ARC from CEH 216 function revealed that the CEH is essential for telomerase RNP function both in vivo and in vitro, 217 consistent with it being required for binding to TERT. Furthermore, our findings are based on 218 data obtained from studying the CEH within two independent circularly permuted telomerase 219 RNA allele contexts (Figures 2 and 3). One of the circular permutations has the RNA ends 220 moved to the distal portion of the template-boundary element, cpTBE, and the other to within 221 the junction in the central hub between the template and pseudoknot, cpJ3 ( Figure 1A). Under 11 both of these different conditions, we found that a 4-bp CEH is sufficient for its function. Fewer 223 base pairs did not permit CEH function, and it is likely that they also do not stably form a helix, 224 providing additional evidence that a paired secondary structure element is necessary at this 225 position within the catalytic core of yeast telomerase RNA.

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As for any functional importance of the sequence of the CEH, our results from sequence-228 randomized, truncated, and extended-helix alleles provide strong evidence that core-enclosing 229 helices with diverse sequences (6 different variations) provide at least basic telomerase activity.

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Functionality of the CEH in these alleles was despite the sensitized context of being tested 231 within miniaturized TLC1 RNA, which, even when otherwise wild-type in sequence, supports 232 rather short telomeres.

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The core-enclosing helix is a conserved feature of telomerase RNAs beyond yeasts, being also 235 present in human, ciliate, and most other telomerase RNAs known to date. Although the CEH 4- . In these species, it has been proposed that TERT simply uses run-245 off reverse transcription, since these RNAs also lack a template-boundary element (Hinkley et 246 al., 1998), so thus differ from yeast and humans. It is worth also noting that the template-12 boundary element and core-enclosing helix appear to be consolidated into a single paired 248 element in human telomerase RNA (Chen and Greider, 2003;Lin et al., 2004). Thus, the CEH in 249 yeast could be an essential TERT-binding site while not performing the orthologous function in 250 some other species. It is likely that the reason for differences with respect to how the RNA and 251 TERT interact is due to the rapid evolution of the RNP.

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Finally, our results also show that binding of TERT to TLC1 is not helix sequence-specific, but