The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

Abstract

Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases. The “bloodstream forms”, which replicate extracellularly in the blood and tissue fluids of mammals, are coated by a monolayer of Variant Surface Glycoprotein (VSG). Switching of the expressed VSG gene is central to parasite pathogenicity because it enables the parasites to evade adaptive immunity via antigenic variation. Adequate levels of VSG expression - 10% of total protein and 7% of mRNA - are attained through very active RNA polymerase I transcription, efficient mRNA processing (trans splicing of a capped leader and polyadenylation), and high mRNA stability. We here show how VSG mRNA stability is maintained. Purification of the VSG mRNA with associated proteins specifically selected CFB2, an F-box mRNA-binding protein that lacks known RNA-binding domains. CFB2 binds to a stabilizing complex (MKT1-PBP1-XAC1-LSM12) that recruits poly(A) binding protein and a specialized cap-binding translation initiation complex, EIF4E6-EIF4G5. The interaction of CFB2 with MKT1 is essential for CFB2’s expression-promoting activity, while the F-box auto-regulates CFB2 abundance via interaction with SKP1, a component of the ubiquitination machinery. The results of reporter experiments indicate that CFB2 acts via conserved sequences in the VSG mRNA 3’-untranslated region. Depletion of CFB2 leads to highly specific loss of VSG mRNA. VSG expression is essential not only for antigenic variation but also for trypanosome cell division. Correspondingly, depletion of CFB2 causes cell cycle arrest, dramatic morphological abnormalities and trypanosome death.

Endoplasmic reticulum-targeting but not translation is required for mRNA balancing in trypanosomes

“…Interestingly, it is completely absent from the genomes of the related African trypanosome species T. congolense and T. vivax, which also have a VSG surface coat. The F-box RNA binding protein (CFB2) has been suggested as a limiting factor which interacts with the 16-mer motif in the VSG 3ʹUTR, thereby actively regulating VSG mRNA abundance (do Nascimento et al, 2020). As an alternative mechanism, VSG mRNA levels might be modulated by a feedback that is dependent on the production of functional GPI-anchored VSG protein (Maudlin et al, 2021).…”

“…This points against a simple counting mechanism. We, therefore, propose that though the interaction between the 16-mer and CFB2 is essential for high expression and stability of VSG mRNA (do Nascimento et al, 2020;Ridewood et al, 2017), the steady-state VSG mRNA amount is regulated at a different level.…”

“…In another recent study an mRNA binding protein CFB2 was found to interact with the 16-mer motif, where it mediates VSG mRNA stability by recruitment of a stabilizing complex. In addition, it was proposed that regulation of CFB2 levels might in turn limit VSG synthesis to avoid excess production (do Nascimento et al, 2020). However, by introducing premature termination codons (PTCs) in the ectopic VSG gene open reading frame (ORF), Maudlin et al reported massively increased total VSG mRNA levels thus questioning whether a direct counting mechanism for VSG mRNA exists (Maudlin et al, 2021).…”

A nuclear enterprise: zooming in on nuclear organization and gene expression control in the African trypanosome

“…The single active VSG gene generates the most abundant protein in the cell (approximately 10% of the total proteome), which results from a combination of high levels of transcription by Pol-I and multiple mechanisms of post-transcriptional control (Navarro and Gull, 2001; Günzl et al ., 2003; do Nascimento et al ., 2020; Viegas et al ., 2020). This renders trypanosomes and their VSG s an amenable model system to study mechanisms underpinning single gene choice, which are not fully understood in any eukaryote.…”