Subunits of RNase P/MRP, co-purify with telomerase and affect telomere length and telomerase in vivo.

Abstract

This thesis is comprised of two projects. The first part has been published and involves epigenetic effects associated with genomic rearrangements in Saccharomyces cerevisiae. My work is a relatively small part of a six-person collaboration. The second part is unpublished, and I designed, conducted, and interpreted all the experiments. Therefore, the abstract is focused on the second part of my thesis work.

The DNA ends of eukaryotic chromosomes are synthesized by the ribonucleoprotein telomerase. Despite its importance in maintaining chromosome stability, telomerase regulation is not completely understood. Recently, components of the RNase P/MRP complexes, Pop1, Pop6, and Pop7 were shown to interact with the RNA component of telomerase TLC1. Here, I used genetic and biochemical approaches in Saccharomyces cerevisiae to understand the function of Pop1, Pop6, and Pop7 on the regulation of telomerase in vivo. Using temperature sensitive alleles of the essential genes that encode Pop1, Pop6, and Pop7, I show that mutants in each gene cause telomere shortening, while mutations in POP1 and POP6 lead to an increase in the abundance of mature TLC1 RNA. I found no difference in the intra-cellular distribution of TLC1 or subunits of telomerase in the mutants. However, TLC1 was less able to associate with two telomerase subunits, Est1 and Est2 and the holoenzyme was defective in telomere binding. Using dimethyl sulfate (DMS) targeted RNA-sequencing, I show that the structure of TLC1 RNA is affected in vivo in the absence of components of RNase P/MRP, while a control RNA was not. The affected regions include the binding sites for the Pop proteins and for Est1 and Est2. These results suggest that the widely conserved and essential Pop1, Pop6, and Pop7 proteins act as chaperones that affect the folding of the scaffold TLC1 RNA. Additionally, the absence of these RNase P/MRP components, prevent the correct assembly of the telomerase holoenzyme and its function in vivo.