Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes

Abstract

24Highly selective gene expression is a key requirement for antigenic variation in several 25 pathogens, allowing evasion of host immune responses and maintenance of persistent 26 infections. African trypanosomes, parasites that cause lethal diseases in humans and 27 livestock, employ an antigenic variation mechanism that involves monogenic antigen 28 expression from a pool of >2500 antigen coding genes. In other eukaryotes, the expression of 29 individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise 30 distal chromosomal loci in the three-dimensional nuclear space. However, trypanosomes lack 31 classical enhancer sequences or regulated transcription initiation and the monogenic 32 expression mechanism has remained enigmatic. Here, we show that the single expressed 33 antigen coding gene displays a specific inter-chromosomal interaction with a major mRNA 34 splicing locus. Chromosome conformation capture (Hi-C), revealed a dynamic reconfiguration 35 of this inter-chromosomal interaction upon activation of another antigen. Super-resolution 36 microscopy showed the interaction to be heritable and splicing dependent. We find that the 37 two genomic loci are connected by the antigen exclusion complex, whereby VEX1 associated 38 with the splicing locus and VEX2 with the antigen coding locus. Following VEX2 depletion, 39 loss of monogenic antigen expression was accompanied by increased interactions between 40 previously silent antigen genes and the splicing locus. Our results reveal a novel mechanism 41 to ensure monogenic expression, requiring the spatial integration of antigen transcription and 42 mRNA splicing in a dedicated compartment. These findings suggest a new means of post-43 transcriptional gene regulation. 44 45 3 Main Text: 46 Monogenic expression, the expression of a single gene from a large gene family, is 47 essential for several important biological processes. One of the most striking examples of such 48 regulation is the expression of a single odorant receptor from more than 1400 genes in 49 mammalian olfactory sensory neurons 1 . Likewise, monogenic expression is a key feature of 50 antigenic variation, an immune evasion strategy used by pathogens such as Plasmodium 51 falciparum or Trypanosoma brucei. Antigenic variation refers to the capacity of an infecting 52 organism to systematically alter the identity of proteins displayed to the host immune system 53 2 . How pathogens ensure the exclusive expression of only one antigen from a large pool of 54 antigen coding genes remains one of the most intriguing questions in infection biology. 55 In T. brucei, a unicellular parasite responsible for lethal and debilitating diseases in 56 humans and animals, 10 million copies of a single variant surface glycoprotein (VSG) isoform 57 are exposed on the surface of the parasite. The exclusive expression of only one VSG gene 58 per cell and the periodic switching of the expressed VSG gene allow the parasite to evade the 59 host immune system and to maintain persistent infecti...

Immunoprecipitation of RNA-DNA hybrid interacting proteins inTrypanosoma bruceireveals conserved and novel activities, including in host immune evasion by antigenic variation

“…We did not detect the other components of the VEX complex (Faria et al, 2019), such as VEX2, however, and so these data may suggest a specific R-loop role of VEX1 (Faria et al, 2021).…”

Functional insights from a surface antigen mRNA-bound proteome

“…For VSG , polymerase I transcription is also necessary in order to produce large amounts of mRNA from a single gene: VSG mRNA comprises at least 5% of total mRNA, and RNA polymerase I transcription from VSG promoters is at least 10 times more efficient than transcription of chromosome-internal regions RNA polymerase II ( Clayton, 2019 ). Moreover, association of the active VSG expression site with the site of spliced leader RNA production results in extremely efficient processing of the VSG mRNA ( Faria et al, 2020 ). However, these measures alone do not suffice: the VSG mRNA is also extremely stable, having a half-life of 1–2 hr compared with less than 20 min for most other mRNAs, including ESAG s ( Ehlers et al, 1987 ; Vanhamme et al, 1999 ; Ridewood et al, 2017 ; Fadda et al, 2014 ).…”

Transcription Dependent Loss of an Ectopically Expressed Variant Surface Glycoprotein during Antigenic Variation in Trypanosoma brucei

“…The recently identified VSG exclusion (VEX) complex, consisting of VEX1 and VEX2, localizes to the active BES in a transcription-dependent manner and defines the single active BES ( 19 , 20 ). The chromatin assembly factor, CAF-1, maintains the epigenetic status of the active BES during DNA replication, and VEX complex association with the spliced leader locus ensures sufficient processing of the VSG mRNA ( 19 – 21 ).…”