IDENTIFICATION OF TbRAP1-INTERACTING FACTORS AND CHARACTERIZATION OF THEIR FUNCTIONS

Abstract

Trypanosoma brucei is a parasite that causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoproteins (VSGs), to evade the host immune response and to establish a long-term infection. VSG genes are located at subtelomeric regions, and the active VSG is transcribed by RNA polymerase I. Antigenic variation is a critical pathogenesis mechanism in T. brucei that has two key aspects: monoallelic VSG expression and VSG switching. Monoallelic VSG expression ensures that at any moment, T. brucei presents a single type of VSG on its cell surface. Telomeres are nucleoprotein complexes located at the chromosome ends. RAP1 is a protein that is conserved from protozoa to mammals and associates with the telomere chromatin. TbRAP1 is essential for T. brucei cell proliferation, telomere/sub- telomere integrity, monoallelic VSG expression, and suppressing VSG switching. To better understand TbRAP1’s essential functions, we attempted to identify TbRAP1’s interaction factors focusing on its conserved functional domains: BRCT, Myb, MybLike (including RRM and DB), and RCT. We identified several proteins that interact with TbRAP1 using TbRAP1 MybLike, Myb to RRM, and RCT domains separately as bait in yeast 2-hybrid (Y2H) screens. First, we identified Importin α (Tb427.06.2640) as a TbRAP1 Myblike domain (aa 633-766)-interacting factor. We also confirmed that TbRAP1 MybLike contains a bipartite nuclear localization signal (NLS) necessary for importing TbRAP1 into the nucleus through interaction with Importin α. Second, we identified many proteins as TbRAP1-Myb-RRM (aa 427-733) interacting candidates, including TbCactin (Tb927.11.11610). Lastly, we also identifiedmany proteins interacting with the TbRAP1-RCT (aa 742-855) domain, including recombinase RAD51 (Tb927.11.2550). Subsequently, we validated the interaction between TbRAP1 and TbCactin by Co-IP and showed that TbCactin is essential for T. brucei proliferation. Cactin, a conserved protein, plays a role in splicing in different organisms and cellular pathways. We examined T. brucei cells' transcriptome before and after TbCactin depletion, highlighting its potential function in trans-splicing. TbCactin depletion led to upregulation of over 300 genes and downregulation of nearly 150 genes, although more analysis is required to determine if TbCactin affects gene splicing. This helps us understand TbCactin’s role in gene expression regulation and cell proliferation.