Identification and function of the VSG transcript-bound proteome

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PROJECT SUMMARY Trypanosoma brucei causes African trypanosomiasis in humans and livestock. In the mammalian infectious Bloodstream-Form (BF) stage, T. brucei expresses a single species of Variant Surface Glycoprotein (VSG) genes from over 2,500 VSG repertoire with a potential of switching to a different VSG gene, a phenomenon known as antigenic variation. This allows the parasite to escape from the action of the host immune system and leads to a chronic infection. The surface of a BF trypanosome cell is coated with about 11 million VSG proteins, which can be translated from about 1,400 copies of VSG mRNA (about 7% of total mRNA). Because VSG is essential for the viability of BF T. brucei, the parasites must maintain the functional high level of VSG mRNA and VSG protein to survive. Almost all genes are transcribed polycistronically in T. brucei, including VSG. One way to achieve the high level of VSG mRNA is through transcription. A single VSG allele is expressed from the transcriptionally active ‘BF Expression Site (BES)’, a Polycitronic Transcription Unit (PTU), containing an RNA pol I promoter, several of Expression-Site Associated Genes (ESAGs) and a VSG. About 15 BESs are present but only one is transcriptionally active and the remaining BESs are repressed. Although high levels of ESAGs and VSG transcripts are produced from the active BES, the abundance differs significantly, with VSG transcripts 100~1000-fold higher than ESAGs. This suggests that there must be trans-acting VSG mRNA specific regulators, most probably RNA-binding proteins (RBPs) that maintain the high level of VSG mRNA. Additionally, VSG is expressed during BF stage but not in other stages of life-cycle during T. brucei differentiation. Despite the importance, almost nothing is known about mechanisms for post-transcriptional regulation of VSG mRNA, except that a short, conserved sequence element embedded into the 3´UTR of VSG mRNA (16-mer) confers both stage specificity and stability. Here we hypothesize that the 3´UTR of VSG mRNA have sequence-specific binding sites for RBPs and that the assembly and composition of this VSG mRNA RiboNucleoprotein Particles, ‘VSG mRNPs’, promote translation and/or protect VSG mRNA from degradation, maintaining the vast amount of VSG mRNA and thus, protein also. In this proposal we aim to identify the components of VSG mRNPs in BF T. brucei. We will first genetically and biochemically identify the components of the VSG mRNP complex in Aim 1 and validate potential candidates in Aim 2. VSG serves multiple roles in the survival of BF trypanosome; it can protect the parasite but can also trigger strong adaptive host immune response. Even though VSG is a strong antigen, developing vaccine therapy for African trypanosomiasis has been difficult due to the antigenic variation. Given that VSG is absolutely essential for BF T. brucei, understanding the VSG expression control mechanisms (specifically how RBPs impact on functions of VSG mRNPs) would not only enable us to discover fundamental biology of T. brucei gene expression but also to discover a new biology that could lead to therapeutic intervention of this human disease. ! !