Authors

Discussion

Widespread polycistronic transcription in parasitic trypanosomatids means that gene expression controls, and adaptation to varying host environments, must operate post-transcription. Consistent with this, a genome-scale massive parallel reporter assay (MPRA) in Trypanosoma brucei identified thousands of regulatory mRNA 3'-untranslated regions (3'-UTRs, PMID: 39285175). After updating annotations for 59% (4,703) of all 3'-UTRs, we found that those associated with increased translation efficiency were enriched in adenine-rich poly-purine tracts (pPuTs), while UTRs associated with increased expression in bloodstream-stage cells were longer than average and also enriched in potentially complementary uracil-rich poly-purine tracts and palindromic sequences. This suggested a ‘zipper hypothesis’ whereby these ‘P5-UTRs’ form secondary structure at lower temperature, and 'unmask' regulatory sequences at increased temperature. Quantification of T. brucei transcriptomes and proteomes following growth at 34°C, 37°C or 40°C revealed a classical heat-shock response, and expression of approx. 1,000 additional transcripts that correlated with temperature, those with P5-UTRs. To assess potential impacts of mRNA secondary structure, we repeated transcriptome analysis in cells lacking the central RNA interference nuclease argonaute (AGO1) and found that thermo-regulation of transcripts with P5-UTRs was abrogated. We have also used genome-wide RNAi screens to connect selected cis-regulatory UTRs to regulatory RNA-binding proteins. In one example involving zinc-sensing, we identified Zinc Nuclear Knuckles (ZNK1, Tb927.11.9510), which is conserved among trypanosomatids and which negatively regulates zinc-transporter mRNA in zinc-replete conditions, and in a 3’-UTR dependent manner.