The oligonucleotide-binding activity of RAP1 is essential for monoallelic VSG expression and cell viability in Trypanosoma brucei

The oligonucleotide-binding activity of RAP1 is essential for monoallelic VSG expression and cell viability in Trypanosoma brucei Amit Gaurav1, Marjia Afrin1, Xian Yang2, Xuehua Pan2, Ranjodh Sandhu1, Yanxiang Zhao2, and Bibo Li1 1 Center for Gene Regulation in Health and Disease, Dept. of Biological, Geo. & Env. Sciences, Cleveland State University, Cleveland, OH 44115 2 Dept. of Applied Biology and Chemical Technology, State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, PRC Trypanosoma brucei is a protozoan parasite that causes fatal African sleeping sickness in humans. While proliferating in the mammalian host, T. brucei stays in extracellular spaces and elicit strong immune responses. However, T. brucei regularly switches its major surface antigen, VSG, thereby effectively evading elimination by the host immune system. VSGs are expressed exclusively from subtelomeric VSG expression sites (ESs), which are large subtelomeric polycistronic transcription units transcribed by RNA Polymerase I. The VSG gene is the last gene in any ES and is located immediately upstream of the telomeric repeats. Although T. brucei has a large VSG gene pool and ~20 nearly identical VSG ESs, only one ES is fully active at any time, resulting in a single type of VSG being expressed on cell surface. We have previously demonstrated that T. brucei RAP1, a telomere protein, is essential for VSG silencing, suppresses the telomeric transcript (TERRA) level, and suppresses DNA recombination-mediated VSG switching events. Additionally, our previous observations indicate that TbRAP1’s function in VSG regulation depends on its association with the telomeric chromatin. Although all known RAP1 homologues associate with the telomeric chromatin, only budding yeast RAP1 has a strong DNA binding activity and directly binds the duplex telomere DNA in vivo through its central Myb and Myb-like domains. We explored the possibility that TbRAP1 also binds telomeric DNA directly. To our great surprise, we found that TbRAP1 has both a DNA and an RNA binding activity in vitro. In addition, TbRAP1 interacts with the active VSG mRNA in vivo. Furthermore, structural analysis indicates that TbRAP1 has both RNA- and DNA-binding domains. Most importantly, mutations that abolish TbRAP1’s DNA or RNA binding activities lead to strong VSG derepression and subsequent growth arrest, indicating that these oligonucleotide-binding activities of TbRAP1 are essential for its cellular functions. The DNA binding activity presumably contributes to TbRAP1’s localization at the telomere. More significantly, the RNA-binding activity is completely novel among all known RAP1 homologues, indicating more drastic evolution of telomere proteins than we have originally understood. Importantly, the discovery of TbRAP1’s RNA-binding activity suggests an unexpected and novel mechanism underlies TbRAP1’s role in silencing subtelomeric genes.