Pedal to the Metal: Nuclear Splicing Bodies Turbo-Charge VSG mRNA Production in African Trypanosomes

Abstract

The African trypanosome Trypanosoma brucei is a parasite of the mammalian bloodstream and tissues, where an antigenically variable Variant Surface Glycoprotein (VSG) coat protects it from immune attack. This dense layer comprised of ∼10⁷ VSG proteins, makes VSG by far the most abundant mRNA (7-10% total) and protein (∼10% total) in the bloodstream form trypanosome. How can such prodigious amounts of VSG be produced from a single VSG gene? Extremely high levels of RNA polymerase I (Pol I) transcription of the active VSG provide part of the explanation. However, recent discoveries highlight the role of pre-mRNA processing, both in maintaining high levels of VSG transcription, as well as its monoallelic expression. Trypanosome mRNAs are matured through trans-splicing a spliced leader (SL) RNA to the 5' end of precursor transcripts, meaning abundant SL RNA is required throughout the nucleus. However, requirement for SL RNA in the vicinity of the active VSG gene is so intense, that the cell reconfigures its chromatin architecture to facilitate interaction between the SL RNA genes and the active VSG. This presumably ensures that sufficient localised SL RNA is available, and not limiting for VSG mRNA expression. Recently, novel nuclear splicing bodies which appear to provide essential trans-splicing components, have been identified associating with the active VSG. These observations highlight the underappreciated role of pre-mRNA processing in modulating gene expression in trypanosomes. Dissecting the function of these nuclear RNA processing bodies should help us elucidate the mechanisms of both VSG expression and monoallelic exclusion in T. brucei.

Keywords: Trypanosoma brucei; antigenic variation; nuclear architecture; nuclear bodies; trans-splicing; variant surface glycoprotein.

FIGURE 1

An assembly of nuclear bodies congregate at the active VSG expression site At the top a bloodstream form (BF) trypanosome is shown with the nucleus (dashed circle) magnified. This contains an assembly of nuclear bodies (coloured circles) at the active VSG expression site (ES), as well as a nucleolus (blue circle). The active ES has a Pol I promoter indicated with a black flag, various Expression site associated genes (ESAGs) with white boxes, and the VSG with a red box (not drawn to scale). The active ES associates with at least one of the spliced leader (SL) RNA gene arrays, shown with individual SL RNA genes (grey boxes) transcribed from Pol II promoters (white flags). Nuclear bodies are shown below as large coloured circles with selected associated protein components indicated. The Expression site body (ESB) (large green circle) at the active ES is associated with a highly SUMO-ylated focus (HSF) (dark green circle). The Spliced Leader Array Body (SLAB) (red circle) associates with the SL RNA gene arrays, of which at least one is associated with the active ES. Two additional nuclear splicing bodies associating with the active ES include the Cajal Body (violet circle) and a novel NUFIP body (purple circle). These nuclear bodies (with the exception of the ESB) are also found in procyclic form T. brucei. The percentage of BF T. brucei cells in G₁ containing one or more of these nuclear bodies is shown in the respective panels [data from (Budzak et al., 2022)].

FIGURE 2

VSG is the mRNA generated at the highest rate in bloodstream form T. brucei. Schematics show the estimated number of mRNA molecules produced per hour from genes in different genomic loci in bloodstream form Trypanosoma brucei. (A) Schematic of an active VSG expression site containing the most abundant mRNA expressed in bloodstream form T. brucei; VSG. The Pol I ES promoter is indicated with a black flag, and high amounts of Pol I transcription with a thick red arrow. Expression Site Associated Genes (ESAGs) and VSG221 are indicated with coloured boxes, pseudogenes with Ψ (grey boxes), and 70 bp repeats with striped boxes. (B) Schematic showing one of the four procyclin loci, which contain the most abundant mRNAs expressed in procyclic form T. brucei. The Pol I promoter is indicated with a black flag, high levels of Pol I transcription with a thick red arrow, and relevant genes with coloured boxes. (C) Schematic of the tubulin locus, which contains the most abundant mRNAs transcribed by Pol II in T. brucei. The upstream Pol II promoter is indicated with a white flag, and low levels of Pol II transcription with a thin red arrow. Three pairs of alternating α-tubulin and ß-tubulin genes (out of a total of eight per locus) are indicated with coloured boxes. For all graphs, the y-axes are the same scale as in panel (A). Values for mRNA molecules generated per hour were derived from Supplementary Tables 1, 2 in Budzak et al. (2022).