brucei transcriptome as well as the identification of heterogeneous mRNA trans-splicing and polyadenylation sites (42–44). Furthermore, RNA-seq has allowed the determination of transcript boundaries

and the detection of potential RNA polymerase II transcription initiation sites at single-nucleotide resolution (43). Transcriptome profiling using a digital gene expression (DGE) approach has also resulted in high-sensitivity detection of differentially expressed genes in different life cycle stages (45,46) and confirmed the existence of differentially expressed gene clusters within the same polycistronic primary transcript units (45). The ChIP-seq (chromatin immunoprecipitation coupled with NGS) approach made possible the mapping of selleck chemical polycistronic transcription units boundaries with greater reproducibility than ChIP-chip (47) and shed light on chromatin-mediated epigenetic controls in trypanosomes (48).

As RNA Palbociclib nmr interference (RNAi) was first described in T. brucei (49,50), it has become a very powerful tool for reverse genetic analyses in African trypanosomes. The first high-throughput systematic RNAi and phenotypic analysis was performed on chromosome 1 genes in T. brucei and documented phenotypes for 30% of the total targeted genes (51). More recent genome-wide RNAi screens in T. brucei revealed a powerful approach for the discovery of drug transporters and activators (52,53). The application of NGS technologies to high-throughput phenotyping with a genome scale RNAi library (RIT-seq) linked thousands of hypothetical genes to essential functions (54). Much of the information generated from T. brucei RNAi analyses can be found in the TrypanoFAN database (http://trypanofan.path.cam.ac.uk/trypanofan/main/) and a tool for the identification of primers for production of RNAi constructs is also publicly available online (http://trypanofan.path.cam.ac.uk/software/RNAit.html). While the genome sequencing of L. braziliensis demonstrated the retention

of an RNAi pathway and confirmed the presence of an RNAi activity in that organism (23), the loss of this pathway in L. major, L. infantum and T. cruzi, among other trypanosomatids, PAK5 clearly results in an inability to exploit this machinery for gene knockdowns. An attempt to reintroduce known RNAi machinery components in T. cruzi genome was not successful (55). However, efforts to knock down the genes in the human host cells have been made at the subgenomic and full genome scale in T. cruzi and are revealing host genes linked to trypanosome intracellular proliferation and survival [B. Burleigh, personal communication and (56)]. Protein translation and turnover are important parts of gene expression regulation. This is particularly the case in trypanosomatids where much of the regulation is believed to occur post-transcriptionally.