Ribonuclease H1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion

Abstract

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination.

Transcriptional initiation of a small RNA, not R‐loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae

“…R-loops are also implicated in Ig class switch, where the RNA G4 aids recruitment of the cytidine deaminase, which leads to recombination (Zhang et al, 2014;Zheng et al, 2015). We predict that other diversity generation systems, many that have G-rich segments associated with them (Smargiasso et al, 2009;Giacani et al, 2012;Walia and Chaconas, 2013;Glover et al, 2013;Beaume et al, 2013;Briggs et al, 2018), may also depend on R-loops and associated G4s to induce programed recombination.…”

“…R-loops are necessary for the initiation of replication in some plasmids and mitochondrial DNA (Xu and Clayton, 1996;Fukuoh et al, 1997). R-loops are also important for T. brucei VSG Av since the loss of RNase HI influenced Av frequencies (Briggs et al, 2018). R-loops are also implicated in Ig class switch, where the RNA G4 aids recruitment of the cytidine deaminase, which leads to recombination (Zhang et al, 2014;Zheng et al, 2015).…”

“…Trypanosoma brucei is the causative agent of African sleeping sickness and utilizes Av to alter a variable surface glycoprotein (VSG) (McCulloch et al, 2015). R-loops form near VSG switch regions and upon deletion of the gene encoding RNase HI, R-loops near VSG are increased and VSG variation is increased (Briggs et al, 2018;Prister and Seifert, 2018).…”

A DOT1B/Ribonuclease H2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation inTrypanosoma brucei

“…Different possibilities for a source of recombination-initiating DNA breaks have been proposed, and a recently published model has suggested the participation of RNA-DNA hybrids (so-called R-loops) (10). R-loops in T. brucei are generated by transcription (11), and usually removed by the two types of Ribonuclease H (RH1 and RH2) (12)(13)(14). DNA lesions and altered VSG expression were observed if R-loops were not processed properly (12,13), or if their formation at the ES was not accurately coordinated by the telomere-associated protein RAP1 (15).…”

“…Proteins associated with this ESB such as the class I transcription factor A (CITFA) complex or the VSG-exclusion (VEX) complex are required for RNA pol I recruitment and monoallelic transcription, respectively (22,23). Interestingly, R-loops also seem to have an impact on transcriptional control; after depletion of monomeric RH1 or the catalytic subunit of trimeric RH2, trypanosomes derepress silent ESs and express different VSGs on their surface (12,13). Furthermore, transcriptional control is also regulated by nucleosome assembly and chromatin remodeling at the promoter region of the ES, mediated by the ISWI (24), FACT (25), CAF-1b, and ASF1A protein complexes (26).…”