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Function of Telomere Protein RAP1 and Telomeric Transcript in Antigenic Variation in Trypanosoma Brucei

Nanavaty, Vishal P
http://rave.ohiolink.edu/etdc/view?acc_num=csu1485424039406009

Abstract Details

2016, Doctor of Philosophy in Regulatory Biology, Cleveland State University, College of Sciences and Health Professions.
Trypanosoma brucei is a parasite that causes fatal African sleeping sickness. Antigenic variation is an obligatory mechanism for 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 switching) or be mediated by DNA homologous recombination (such as gene conversion and reciprocal DNA crossover/telomere exchange). However, regulation of VSG switching is poorly understood. We previously found that T. brucei RAP1, a telomere protein, is essential for silencing subtelomeric VSG genes. Here we found that transient depletion of TbRAP1 increases the VSG switching frequency, and most switchers in TbRAP1-depleted cells arose thorough VSG-associated gene conversion events. Also, we detected increased amount of DSBs in the active and silent ESs upon TbRAP1 depletion. However, the underlying mechanisms of how TbRAP1 suppresses DSBs at telomeres/subtelomere remained unknown. T. brucei telomeres are transcribed, generating long, non-coding RNA called TERRA (Telomeric repeat-containing RNA). Now, we found that depletion of TbRAP1 not only leads to derepression of telomeric silent VSGs, but also results in increased TERRA levels. In addition, we observed a sixteen-fold increase in telomeric RNA:DNA hybrids (R-loops) in TbRAP1-depleted cells. R-loop has been shown to induce DSBs in yeast, mouse, and human cells and are sensitive to RNaseH, a ribonuclease that cleaves the RNA strand of the RNA:DNA hybrid. Ectopic expression of TbRNaseH1 in TbRAP1 RNAi cells resulted in reduction of the telomeric R-loop levels and reduced the DSB amount 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, resulting in increases VSG switching frequency. We have also identified that transcription read-through into the telomere repeats from the adjacent active ES (but not from silent ESs) contributes to TERRA synthesis.
Bibo Li, Ph.D. (Advisor)
Valentine Boerner, Ph.D. (Committee Member)
Aaron Severson, Ph.D. (Committee Member)
Michelle Longworth, Ph.D. (Committee Member)
Sailen Barik, Ph.D. (Committee Member)
Derek Taylor , Ph.D. (Committee Member)
249 p.
Biology; Genetics; Molecular Biology
Trypanosoma brucei; VSG switching; TERRA; R-loop; telomeric recombination; RAP1; DSB

Recommended Citations

Citations

  • Nanavaty, V. P. (2016). Function of Telomere Protein RAP1 and Telomeric Transcript in Antigenic Variation in Trypanosoma Brucei [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1485424039406009

    APA Style (7th edition)

  • Nanavaty, Vishal. Function of Telomere Protein RAP1 and Telomeric Transcript in Antigenic Variation in Trypanosoma Brucei. 2016. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1485424039406009.

    MLA Style (8th edition)

  • Nanavaty, Vishal. "Function of Telomere Protein RAP1 and Telomeric Transcript in Antigenic Variation in Trypanosoma Brucei." Doctoral dissertation, Cleveland State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1485424039406009

    Chicago Manual of Style (17th edition)

csu1485424039406009
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© 2016, all rights reserved.
This open access ETD is published by Cleveland State University and OhioLINK.