Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

Telomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae , the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm 7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. Here, we first show that Sm 7 can perform this function 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 tested the hypothesis that the location of the Sm 7 -binding site relative to the 3′ end is contrastingly important. When we moved the Sm site to locations 5′ of its native position, we observed that this stabilized shorter forms of poly(A)– TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm 7 binding to TLC1 controls where the mature poly(A)– 3′ end is formed. In summary, our results show that Sm 7 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 Sm 7 in telomerase RNA biogenesis.