Quantification of Est1, Trt1 and TER1: The Protein Subunits and RNA Template that Comprise the Schizosaccharomyces pombe Telomerase Holoenzyme

Abstract

Telomerase reverse transcriptase provides a solution to the “end-replication problem” in most eukaryotes through elongation of the G-rich 3’ overhang at the ends of the linear chromosomes, known as telomeres, which distinguish natural chromosomal ends from DNA double strand breaks and prevent aberrant activation of the DNA damage checkpoint. Previous characterization of the telomerase holoenzyme in Schizosaccharomyces pombe has identified the two protein subunits Est1 and Trt1, as well as the RNA template termed TER1. The precise cellular concentration of homologues of these components has been quantified in other organisms including Saccharomyces cerevisiae. However, their quantity in S.pombe cells remains unknown. This series of experiments seeks to determine the absolute abundance of each of the S. pombe telomerase holoenzyme components per cell in vivo. We quantified the Est1 and Trt1 protein subunits containing a 13Myc epitope tag using western blot assays followed by comparison to a standard 13Myc-tagged protein of known concentration. We determined that there are 66.9±18.6 molecules of Est1 per cell and 47.9±8.5 molecules of Trt1 per cell. TER1 quantity, determined using reverse transcription qRT-PCR and comparison to a standard curve of in vitro transcribed RNA, was found to be present at ~17 molecules per cell. Increased copy numbers of the telomerase holoenzyme components have been suggested to allow cancer cells to evade the normal confines of cell proliferation. Thus, conclusions reached regarding copy numbers may have significant implications in our understanding of the role of the protection of S. pombe telomere ends and suggest models of applicability across evolutionarily divergent organisms.