Significance: Telomerase, which extends DNA at chromosome ends, is composed of an RNA template and a catalytic protein subunit, telomerase reverse transcriptase (TERT). TERT gene expression has been of great interest because it is required for proliferation of most cancers, but expression investigations have been limited due to low endogenous mRNA levels. Here, we employ single-molecule RNA fluorescent in situ hybridization (FISH) in 10 human cancer cell lines and make findings that are unanticipated based upon bulk TERT mRNA measurements. For example, there is great cell-to-cell variation in the number of transcription sites, and spliced TERT mRNA has greater nuclear localization than cytoplasmic. Overall, our findings provide insights regarding TERT expression, localization patterns, and variability in cancer on a single-cell level.

Telomerase RNA contains a template for synthesizing telomeric DNA and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase RNA, TLC1, is bound by the Sm₇ protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. However, it remains unclear (1) whether Sm₇ retains this function when its binding site is repositioned within TLC1, as has been shown for other TLC1-binding telomerase subunits, and (2) how Sm₇ stabilizes poly(A)– TLC1. Here, we first show that Sm₇ can stabilize poly(A)– TLC1 even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we show that when an Sm site is inserted 5′ of its native position and the native site is mutated, Sm₇ stabilizes shorter forms of poly(A)– TLC1 in a manner corresponding to how far upstream the new site was inserted, providing strong evidence that Sm₇ binding to TLC1 controls where the mature poly(A)– 3′ is formed by directing a 3′-to-5′ processing mechanism. In summary, our results show that Sm₇ and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm₇ in telomerase RNA biogenesis.

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-ntSaccharomyces cerevisiaetelomerase 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 activein vitroand to maintain yeast telomeresin 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 andin vivocomplementation 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 amongSaccharomycesspecies, 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.