Talk on Saturday 11:15-11:30am submitted by Vishal Nanavaty
Trypanosoma brucei RAP1 suppresses VSG switching by suppressing levels of telomeric transcript levels and RNA:DNA hybrids
Vishal Nanavaty (Biological, Geological and Environmental Sceinces, Cleveland State University), Ranjodh Sandhu (Department of Microbiology and Genetics, University of California, Davis), Sanaa Jehi (Department of Molecular and Experimental Medicine, The Scripps Research Institute), Unnati Pandya (Department of Medicine, Division of Translational Medicine, New York University School of Medicine), Bibo Li (Biological, Geological and Environmental Sceinces, Cleveland State University)
Telomeres are the nucleoprotein complexes present at linear chromosome ends. They protect natural chromosome ends from illegitimate repair and recombination, maintain a proper length for continued cell proliferation, and silence subtelomeric genes. Despite of having a heterochromatic structure, telomeres are transcribed into long non-coding RNAs called TERRA in many organisms. In yeast and human cells high levels of TERRA expression has been shown to increase the amount of RNA:DNA (R-loop) hybrids at the telomere, which in turn increases homologous recombination (HR) locally. It was previously shown that in Trypanosoma brucei, a parasite causing the fatal sleeping sickness in humans, telomeres are transcribed. However, T. brucei TERRA functions are unexplored. Antigenic variation is an obligatory mechanism for a long-term survival of T. brucei inside its mammalian host. T. brucei expresses VSG as its major surface antigen and regularly switches its VSG coat to evade the host immune response. Although T. brucei genome has more than 2,500 VSG genes and pseudogenes, it only expresses one VSG from one of 15 subtelomeric VSG expression sites (ESs) at any time. VSG switching can be transcriptional (in situ switch) or DNA HR-mediated (gene conversion or reciprocal DNA crossover). We previously found that T. brucei RAP1, a telomere protein, is essential for silencing subtelomeric VSGs. Here we found that a transient depletion of TbRAP1 increases the VSG switching frequency, and the majority of the switchers arose through HR-mediated VSG gene conversion. We also observed increases in TERRA levels and telomeric RNA:DNA hybrid amounts in TbiRAP1 depleted cells. R-loops are sensitive to RNAseH, which cleaves the RNA strand of the DNA:RNA hybrid. Ectopic overexpression of RNaseH1 in TbRAP1 RNAi cells resulted in reduction of the telomeric R-loops back to the WT level and suppressed the elevated VSG switching frequency phenotype. Therefore, we propose that increased TERRA and R-loop levels in TbRAP1-depleted cells mediate DSB-induced VSG gene conversion and VSG switching.
Keywords: Telomere Transcript - TERRA, RNADNA hybrids, VSG switching