Novel Telomerase RNA Gain‐of‐Function Variants in Saccharomyces cerevisiae

Abstract

Telomerase is a ribonucleoprotein enzyme complex that maintains telomeres at the ends of linear eukaryotic chromosomes. Telomerase minimally requires a telomerase reverse transcriptase (TERT) protein that uses a non-coding telomerase RNA as a template to extend 3ʹ ends of lagging strands. In addition to binding TERT and providing a template, telomerase RNA plays additional roles in binding accessory proteins and contributing to catalysis. Telomerase RNAs are rapidly evolving in length, sequence and structure, making phylogenetic comparisons challenging and limiting our understanding of telomerase structure-function relationships. Much of our current grasp of telomerase RNA function results from of loss-of-function mutations. In order to better understand telomerase RNA function, we devised a novel screening strategy to identify gain-of-function alleles in the budding yeast telomerase RNA. Briefly, our screen utilizes a counter-selectable marker, URA3, in a subtelomeric region that is sensitive to telomere position effect. When telomeres are long, the URA3 gene will be silenced to a greater extent, allowing better growth of yeast on media containing the chemical 5-fluoroorotic acid (5-FOA). To identify novel mutations, we used error-prone PCR to generate a library of random mutations in a miniaturized version of telomerase RNA, Mini-T(460). A pilot screen of our mutant library has isolated 5 new gain-of-function alleles that allow increased growth of yeast in the presence of 5-FOA. We are currently scaling up our screen to identify additional gain-of-function alleles. Simultaneously, we are investigating the mechanism by which these telomerase RNA alleles lead to longer telomeres. We hypothesize that these mutations could act through one or more mechanisms, including increasing RNA abundance, RNA stability, catalysis, repeat-addition processivity, and/or protein binding. Together, our work will significantly increase understanding of the structure and function of telomerase RNA.