The S. cerevisiae telomerase RNP: From assembly to action

[摘要] Telomeres, the protective end-caps of linear eukaryotic chromosomes, progressively shorten as cells divide and, in most eukaryotes, are lengthened by the ribonucleoprotein enzyme telomerase. The telomerase RNA — one of the two core subunits of telomerase — contains the template for reverse transcription of new telomeric repeats and serves as a scaffold for the telomerase RNP. In Saccharomyces cerevisiae, the telomerase RNA (TLC1) tolerates repositioning of several of its protein-binding sites and deletion of the sequences in between, suggesting that TLC1 acts as an organizationally flexible scaffold that need only tether together the various subunits of telomerase.In Saccharomyces cerevisiae, telomerase is recruited to telomeres through two pathways. While the primary recruitment pathway has been studied extensively, the secondary pathway, which involves the Ku70/Ku80 heterodimer bound to TLC1, has remained poorly understood. I found that TLC1-bound Ku recruits telomerase to telomeres by binding to the telomeric silent chromatin protein Sir4. I also found that this pathway is inhibited by the negative regulators of telomerase, Rif1 and Rif2, which compete with Sir4 for binding to the telomere-binding protein Rap1. My research suggests that the Ku-mediated recruitment pathway serves a regulatory function and connects the Rif proteins with the primary telomerase-recruitment pathway. TLC1 is also bound by the Sm7 complex, which stabilizes the major isoform of TLC1. While the other TLC1-bound holoenzyme subunits, Est1 and Ku, retain their functions when repositioned in full-length TLC1, this has not been tested for Sm7. By repositioning the Sm-site via circular permutation, I found that Sm7 retains function at diverse positions within TLC1, suggesting that all RNA-bound subunits of telomerase are organizationally flexible modules. I also used Sm-site repositioning without circular permutation to show that Sm7 defines the mature 3ʹ end of the major TLC1 isoform. Lastly, I developed a novel technique, CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid, which combines the yeast two-hybrid assay with the Streptococcus pyogenes CRISPR machinery to study RNA-protein interactions. I found that CARRY two-hybrid can detect RNA-protein interactions with high specificity and sensitivity, and I used this assay to investigate regions of the TLC1 core required for binding TERT, the catalytic subunit of telomerase.

[发布机构] Johns Hopkins University

[效力级别] telomere

[关键词] telomerase;telomere;yeast;RNA;CRISPR;Biology