Biochemical and functional investigation of TbRAP1 and its role in T. brucei variant surface glycoprotein regulation

Abstract

Protozoan parasite Trypanosoma brucei (T. brucei) causes African sleeping sickness in humans and Nagana in cattle. These diseases are difficult to cure because T. brucei can evade the host immune response by regularly switching its major surface antigen, Variant Surface Glycoprotein (VSG). T. brucei has a repertoire of over 2,000 VSG genes. These genes are expressed from one of the Expression Sites (ESs) located immediately upstream of telomeres in a strictly monoallelic manner. Previous studies have found that telomeric proteins belonging to the Shelterin complex play important roles in regulating VSG monoallelic expression and switching. Specifically, TbRAP1, a member of the telomere Shelterin complex, is essential for repressing the expression of VSGs located in "silent" ESs. We have carried out biochemical and structural studies of TbRAP1 to elucidate the molecular mechanism of TbRAP1-mediated VSG silencing. First of all, we identified an unusual DNA binding activity in TbRAP1 MybLike domain (TbRAP1-ML) by in vitro EMSA assay and NMR titration experiments. A positively charged 737RKRRR741 patch (R/K patch) within the MybLike domain is responsible for electrostatics-based, sequence non-specific binding to both single- and double-stranded DNA. Consequently, we have termed TbRAP1 (aa 734-761) containing the R/K patch as DNA binding (DB) region. Cell-based assays reveal that this electrostatics-based DNA binding activity is essential for TbRAP1's telomere localization, VSG silencing, telomere integrity, and cell proliferation. Secondly, we also determined the structure of TbRAP1 MybLike domain (aa 639- 761) by NMR. Unexpectedly, this domain turned out to contain a canonical RNA Recognition Module (RRM) with the characteristic topology of a four-stranded anti-parallel β-sheet plus two α-helices. Structural superimposition and sequence alignment of TbRAP1 RRM domain to other classic RRMs suggest that this module may have potential RNA binding activity. To validate whether the TbRAP1 RRM domain possesses RNA binding activity, we used EMSA and NMR titration methods to assess the interaction between TbRAP1 RRM and various RNA substrates. Our results show that the RRM domain binds preferably to VSG mRNA. And the conserved F655 and F694 residues located on the canonical RNA binding site are critical for this interaction. Additionally, the positively charged DB segment also showed electrostatics-based non-specific RNA binding. Given that no RAP1 homologs have been reported to possess RNA binding activity, our finding of TbRAP1's RNA binding activity through its RRM domain is completely novel. Our research team also carried out in vivo studies to delineate the functional significance of the RNA binding activity in TbRAP1 RRM domain. Indeed, TbRAP1 associates with the active VSG mRNA in vivo as confirmed by RNA-IP experiments. Additionally, mutations that abolish TbRAP1's RNA binding activity led to the expression of all VSGs located in silent ESs cell growth defect. These data indicate that TbRAP1's RNA binding activity is essential for VSG monoallelic expression and cell viability. Most importantly, mutations that abolish TbRAP1's RNA binding activity caused a decrease in the mRNA level of the only active VSG. Thus, the novel RNA-binding activity mediated by TbRAP1 RRM module may be essential for monoallelic expression of VSG by sustaining the high expression level of the only active VSG while silencing the rest. In summary, using a combination of biochemical, structural, and functional studies, my thesis work has uncovered novel DNA- and RNA-binding activities in TbRAP1. The electrostatics-based sequence non-specific DNA binding activity mediated by the DB region is critical for telomere localization of TbRAP1 and VSG silencing. The novel RNA binding activity mediated by the TbRAP1 RRM domain is essential for sustaining monoallelic expression of the only active VSG.