RNA connectivity requirements between conserved elements in the core of the yeast telomerase RNP

Abstract

Telomerase is a specialized chromosome end-replicating enzyme required for genome duplication in many eukaryotes. An RNA and reverse transcriptase protein subunit comprise its enzymatic core. Telomerase is evolving rapidly, particularly its RNA component. Nevertheless, nearly all telomerase RNAs, including those of H. sapiens and S. cerevisiae, share four conserved structural elements: a core-enclosing helix (CEH), template-boundary element, template, and pseudoknot, in this order along the RNA. It is not clear how these elements coordinate telomerase activity. We find that although rearranging the order of the four conserved elements in the yeast telomerase RNA subunit, TLC1, disrupts activity, the RNA ends can be moved between the template and pseudoknot in vitro and in vivo. However, the ends disrupt activity when inserted between the other structured elements, defining an Area of Required Connectivity (ARC). Within the ARC, we find that only the junction nucleotides between the pseudoknot and CEH are essential. Integrating all of our findings provides a basic map of functional connections in the core of the yeast telomerase RNP and a framework to understand conserved element coordination in telomerase mechanism.

Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

“…Structural characterization of the S. cerevisiae telomerase RNA, TLC1, has been challenging due to its large size, the rapid structural evolution of even essential core RNA elements and their considerable mutational tolerance Lin et al 2004;Zappulla et al 2005;Lebo and Zappulla 2012;Mefford et al 2013). A range of secondary-structure models have been proposed for the essential pseudoknot within the catalytic core (Supplemental Fig.…”

“…These regions may introduce considerable physical flexibility in the core, particularly J3, which is 16 nt long and tolerates a wide variety of mutations (Mefford et al 2013). However, both J1 and J2 are comparatively less reactive (Fig.…”

“…In Saccharomyces cerevisiae, all accessory protein binding sites can be repositioned with retention of function in vivo, illustrating that the 1157-nt yeast telomerase RNA, TLC1, is a flexible scaffold (Zappulla and Cech 2004;Zappulla et al 2011;Mefford et al 2013).…”

“…Despite extensive divergence of telomerase RNA in sequence and length (spanning from 147 nt in a ciliate species to >2 kb in some fungi), most telomerase RNAs share four common structural elements within the central catalytic core, including the template, a template-boundary element, a core-enclosing helix, and a catalytically important pseudoknot (Lin et al 2004;Mefford et al 2013). In the core of TLC1, the elements are functionally linked by an Area of Required Connectivity (Mefford et al 2013).…”

“…In the core of TLC1, the elements are functionally linked by an Area of Required Connectivity (Mefford et al 2013). The high conservation of telomerase RNA core structures in spite of rapid evolution of the RNA overall suggests that the cores function similarly.…”