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

Abstract

The cell surface of bloodstream form African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). By continuously changing the expressed VSG antigen, the parasites can survive the host’s immune response. The VSG is highly expressed in Trypanosoma brucei, accounting for approximately 10 – 20% of total mRNA. Depletion of VSG mRNA is lethal, and a counterbalancing of the mRNA levels occurs when two or more VSGs are simultaneously expressed. How the VSG expression levels are regulated is unknown. Here, by using inducible and constitutive systems for ectopic VSG expression, we have discovered that (i) the endogenous VSG mRNA level is downregulated only when the ectopic VSG is targeted to the ER, (ii) VSG translation is dispensable and in fact, (iii) the regulation of VSG mRNA levels does not depend on a VSG open reading frame. We propose that feedback elicited at the ER regulates the VSG mRNA amounts to avoid overshooting the secretory pathway capacity. In this way, VSG expression is quantitatively and qualitatively fine-tuned. Balancing the overall number of ER-targeted mRNAs could well be a general mechanism in cell biology. The trypanosome system with just one dominant mRNA species provides a versatile model for studying this phenomenon.

The dual role of the 16mer motif within the 3’ untranslated region of the variant surface glycoprotein ofTrypanosoma brucei

“…The double-expressor cell lines 221 ES 121 WT and 221 ES 121 N46-48 were generated by transfecting the plasmids pbRn6.M1.6 WT and pbRn6M1.6 TGA46-48ACT.nPPT , both linearised with SacI and SalI into T. brucei 13-90 cells, respectively. The plasmid pbRn6.M1.6 WT was generated as described by (Aroko et al ., 2021). pbRn6M1.6 TGA46-48ACT.nPPT on the other hand was generated by mutagenesis PCR using the fusion PCR approach.…”

“…The pES plasmids were linearised with AvrII and KpnI and transfected into T. brucei 13-90 cells to produce the VSG121 double-expressor cell lines (WT, [46][47][48][49][50][51][52][49][50][51][52][53] into T. brucei 13-90 cells, respectively. The plasmid pbRn6.M1.6 WT was generated as described by (Aroko et al, 2021). pbRn6M1.6 TGA46-48ACT.nPPT on the other hand was generated by mutagenesis PCR using the fusion PCR approach.…”