The role of genomic location and flanking 3′UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

Abstract

Summary Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I‐transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16‐mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I‐transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell‐cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell‐cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I‐transcribed ES, as well as conserved VSG 3′UTR 16‐mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei.

Dynamic regulation of the Trypanosoma brucei transferrin receptor in response to iron starvation is mediated via the 3’UTR

“…Under iron starvation conditions the repressive effect of the ESAG6 3’UTR is reduced, resulting in an increase of expression that is most likely driven by an increase in transcript abundance, as Tb TfR increases equally at the mRNA and protein level (9, 10). The involvement of the 3’UTR in iron starvation response suggest that it contains secondary structural elements or sequence motifs that are recognised by RNA binding protein(s) (RBPs), but the high level of sequence conservation in the UTR makes it likely that any such features are more extensive than the 16-mer regulatory sequence identified in VSG mRNAs (28). Further experiments will be required to identify any regulatory features present in the ESAG6 3’-UTR, and whether they have a positive or negative regulatory role.…”

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

“…CFB2 depends on a conserved 16mer in the 3'-UTR Supplementary Fig 10). Experiments using reporter mRNAs have shown that the 16-mer is required for high mRNA abundance and stability in bloodstream forms (12,13). It is also required for m 6 A modification of the poly(A) tail, which plays a role in VSG mRNA stabilization (67).…”

“…The 16mer sequence is required to maintain the ~2h VSG half-life in bloodstream forms (11)(12)(13). After uptake into the tsetse fly vector, the trypanosomes convert to the procyclic form; VSG transcription is shut off, the mRNA becomes unstable (11,12) and the VSG coat is lost.…”

Evolution of Antigenic Variation in African Trypanosomes: Variant Surface Glycoprotein Expression, Structure, and Function

“…Another mechanism for elevating VSG production involves post‐transcriptional regulation. VSG mRNAs are exceptionally stable ( t 1/2 ≈2 h), a phenomenon mediated by a conserved 14‐mer in the 3′ UTR . Mutation of the 14‐mer reduces stability (≈5.5‐fold) to levels equivalent to most other mRNAs.…”