Poster abstracts
Poster number 129 submitted by Samantha Sanford
Telomerase Regulation in Pathogenic Protozoa
Samantha Sanford (University of Pittsburgh Graduate School of Public Health, Infectious Disease and Microbiology), Priscilla Wong (Carnegie Mellon University, Department of Chemistry, CNAST), Kausik Chakrabarti (Carnegie Mellon University, Department of Chemistry, CNAST)
Abstract:
Telomere maintenance is the major physiological means of unlimited cellular proliferation, especially in rapidly dividing pathogenic eukaryotes such as Trypanosoma brucei and Plasmodium falciparum. Stable telomeres have the ability to protect the chromosome termini from degradation and recombination, which is critical for virulence and proliferation of the pathogen. In order to keep the telomere length stable, the synthesis of telomeric DNA by telomerase is necessary. Telomerase is a ribonucleoprotein complex which adds a telomere repeat sequence to the 3’ end of telomeres. It contains a telomerase reverse transcription enzyme (TERT) and carries its own RNA molecule, (TR), which is used as a template when it elongates. The objective of this project is to determine the structural requirements for TR-TERT interactions. Compared to human and yeast models, T. brucei TR has different RNA-protein interactions and conformational changes which could result in different telomerase activity(1). Using SHAPE, the RNA structure and folding is determined through a comparison of several different mutations of various regions including the template and pseudo knot domain, as well as the 3’ and 5’ CD Box domains. The mutant RNA was in vitro transcribed and chemical foot printing was used to determine the RNA structure by SHAPE analysis (2) To determine if the substrate binding of the RNA is affected by any mutation that is in the template gel shift assays will be done. To explore how this structural component interacts with chromatin, preliminary results in Plasmodium have shown that a histone deacetylase, SIR2A, may have the ability to coordinate telomere, telomerase, and subtelomere. By studying the chemical structure and properties of this domain, we will better understand how it can affect the telomerase enzymatic activity properties.
References:
1. Sandhu R, Sanford S, Basu S, Park M, Pandya UM, Li B, Chakrabarti K. 2013. A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei. Cell Res 23:537-551.
2. Wilkinson KA, Merino EJ, Weeks KM. 2006. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1:1610-1616.
3. Turner R, Shefer K, Ares M, Jr. 2013. Safer one-pot synthesis of the 'SHAPE' reagent 1-methyl-7-nitroisatoic anhydride (1m7). RNA 19:1857-1863.
Keywords: Telomerase, RNA Structure, Histone Deacetylase