DescriptionTalk: “Multi-allelic exclusion by an allele-selective RNA-DNA helicase in African trypanosomes”
Authors: Joana Correia Faria1#, Hideharu Hashimoto2, Michele Tinti1, Catarina de Almeida Marques1§, Erik W Debler2, David Horn1
Large gene-families often exhibit monogenic expression, with contingent processes including antigenic variation for immune evasion in parasites, and olfaction and B-cell development in mammals. Despite intense study, the mechanisms that underpin these paradigmatic examples of stochastic gene choice and exclusion remain somewhat mysterious.
Substantial recent progress yielded Variant-Surface-Glycoprotein (VSG) exclusion factors in African trypanosomes and a new appreciation of the context in which they operate. Specifically, VSG-exclusion-2 (VEX2) accumulates at the active-VSG expression-site and binds VEX1 at a trans-splicing locus on another chromosome; other VSGs are excluded from this sub-nuclear expression-factory (PMIDs: 31289266; 33432154).
We performed single-cell-RNA-Seq following VEX2 depletion, revealing a surprisingly complex mixture of simultaneously active VSGs in single cells. Notably, we also observed a consistent hierarchy of VSG derepression.
ChIP-Seq indicated strong enrichment of VEX2, which forms a native complex of ∼1 MDa, at the active-VSG expression-site. Using super-resolution microscopy, VEX2 N- and C-termini were distinctively visualised extending towards the active-VSG and the splicing locus, respectively, revealing an allele-selective inter-chromosomal bridge.
Further, a recombinant predicted helicase core from VEX2 displayed RNA-DNA-helicase activity in vitro. To assess whether this activity was required for VSG-exclusion, we established a CRISPR/Cas9-mediated saturation-mutagenesis assay using FACS followed by amplicon-Seq profiling. Replacement of a critical amino acid with any other amino acid disrupted allelic exclusion.
Finally, we found that the VEX1 N-terminus interacts with VEX2, stabilising it and sustaining exclusion.
This work begins to reveal the mechanisms by which the VEX complex promotes stochastic VSG gene choice and allelic exclusion in African trypanosomes.
|Period||13 Sept 2022 → 17 Sept 2022|
|Location||Woods Hole, United States|
|Degree of Recognition||International|