Authors

L Lansink²; M Jones²; AA Dowle¹; J Correia Faria³;  ¹ University of York, UK;  ² Department of Biology, University of York, UK;  ³ Biology Department & York Biomedical Research Institute, University of York, UK

Discussion

African trypanosomes use antigenic variation to evade the host immune response. They hide by obscuring invariant membrane-bound antigens under a dense coat of one out of >2000 Variant Surface Glycoprotein (VSG) isoforms. Their ability to express a single VSG at any given time, monogenic expression, is essential for successful antigenic variation. However, the molecular mechanisms underpinning this complex process are not fully understood.

In Trypanosoma brucei bloodstream-form, the single active-VSG is transcribed by RNA-Polymerase I within the expression-site body (ESB). In spatial proximity to the ESB are other subnuclear bodies including the Spliced-Leader-(SL)-associated body (SLAB), essential for trans-splicing, and the NUFIP ‘body’, also thought to play a role in splicing. This spatial integration of transcription and splicing facilitates mRNA maturation and combined with other mechanisms enables the production of vast amounts of the active-VSG (10% of the total proteome).

VSG-Exclusion Protein 2 (VEX2) specifically localises to the ESB and establishes an inter-chromosomal bridge via VEX1 to the SL-locus; other VSGs are excluded from this sub-nuclear ‘transcription and splicing factory’.

To define the protein network within the ESB and spatially proximal bodies and better understand the sub-nuclear context in which VEX1 and VEX2 operate, we fused VEX1-C, VEX2-N and VEX2-C with TurboID and performed proximity labelling. Proteomic analysis of proximally enriched proteins captured known interactors (VEX2, VEX1 and CAF-1 subunits), as well as components of the ESB and neighbouring bodies that have been previously identified, therefore validating our approach. Notably, we identified novel components of the ‘transcription and splicing factory’, and their localisation has been subsequently validated using super resolution microscopy. Among these were three novel ESB-specific components, a range of ESB-enriched proteins and one new SLAB component as well as four NUFIP enriched proteins. We are currently functionally characterising the novel ESB-specific components, two of which are stage-specific, and defining their very own spatial interactome.

Following the discovery of the VEX proteins and ESB1, the identification of novel ESB-specific factors using proximity labelling constitutes a significant advance in the understanding of the molecular mechanisms governing the biology of antigenic variation regulation in African trypanosomes.