A trans- splicing model for poly(A)-tail addition during VSG mRNA maturation in Trypanosoma brucei

Trypanosomes are characterized by polycistronic transcription and a pre-mRNA processing pathway in which spliced leader (SL) trans -splicing and cleavage and polyadenylation produce individual mature mRNAs. SL and poly(A)-tail addition within the intergenic regions are obligatorily coupled, yet the molecular basis for this coupling has remained unknown. Here we identified a splicing enhancer sequence required for efficient maturation of the highly abundant variant surface glycoprotein (VSG) mRNA and showed that it recruits factors implicated in RNA processing and nuclear export, including the conserved RNA helicase Sub2. RIP-seq revealed that Sub2 associates not only with VSG pre-mRNA but also with SL RNA precursors carrying long genome-encoded 3' extensions. Integration of these findings with analyses of published U1C iCLIP and cold shock domain protein 1 (CSD1) seCLIP datasets supports a model in which polyadenylated SL RNA precursors, rather than mature SL RNPs, serve as substrates for trans -splicing. We propose that VSG pre-mRNA forms an integral component of a specialized spliceosomal assembly that catalyzes successive rounds of SL and poly(A)-tail trans -splicing, driven by extensive base-pairing interactions. This model provides a mechanistic framework for the strong coupling of mRNA 5'- and 3'-end formation in trypanosomes and suggests that spliceosome-catalyzed polyadenylation represents an evolutionarily ancient strategy for eukaryotic pre-mRNA processing. More broadly, it challenges the notion that the two transesterification steps of RNA splicing require a universally conserved arrangement of spliceosomal RNA components.