Unicellular flagellated trypanosomatid parasites are responsible for various life-threatening diseases in humans and major production losses (e.g., meat, milk, and fertility) in animals. These parasites have a complex life cycle, shuttling between mammalian host and fly vector. To respond and adapt to these extremely variable environments, trypanosomatids need to finely tune the expression level of hundreds of genes. In trypanosomatids, surprisingly, functionally often unrelated genes are transcribed simultaneously in polycistronic units. Evidence suggest post-transcriptional events are the main mechanism of gene regulation in trypanosomatids, controlling the transcripts in terms of processing, localization, stability, and translation. Combinatorial, cooperative as well as competive, interactions among RNA regulatory elements (RREs) play a major role in this process by recruiting RNA binding proteins (RBPs) to their target RNAs.

Despite of paramount importance, high resolution mapping of RREs in the transcriptome have proved a challenging task, mainly due to the characteristics of RREs, including their small length (normally less than 10nt), high degeneracy, and high dependence on the residing context for the functionality. Here, taking advantage of the current knowledge on the trypanosomatid gene-expression mechanisms, we developed a functional genomics approach that combines flow cytometry with high-throughput sequencing techniques to simultaneously examine the regulatory role of a large number of short sequences in vivo. To capture the regulatory role of the candidate short sequences in either RNA stability and/or translation, our strategy utilises a dual-color reporter system expressing enhanced GFP (eGFP) that accepts the short sequences in its 3’-UTR region and mCherry protein that acts as the internal control. Doing so, we expect that insertion of a stabilizer or translational-enhancer element increase the ratio of GFP-to-mCherry, while insertion of a destabiliser or translational suppressor decreases this ratio. To decipher the regulatory roles, after the stable transfection of the library, cells were categorized based on their GFP-to-mCherry ratio and then subjected to high-throughput sequencing.