The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. cause the major human diseases African Sleeping Sickness, Chagas' disease, and Leishmaniasis, respectively. Since drugs for these neglected tropical diseases are limited, and, in part, toxic and difficult to administer, and since parasite resistance to these drugs is on the rise, it becomes increasingly important to develop new strategies of chemotherapeutic intervention. Our research has focused on trypanosome gene expression because the underlying mechanisms deviate substantially from those in the human host. For example, polycistronic transcription of protein coding genes and processing of pre-mRNA by spliced leader (SL) trans splicing are parasite-specific steps in mRNA synthesis and maturation. We have discovered that depletion of T. brucei cyclin-dependent kinase CRK9 (cdc2-related kinase 9) not only disturbed mitosis and cytokinesis as previously reported, but also led to a massive trans splicing block. In addition, we observed that ablation of this kinase caused the complete loss of phosphorylation of the largest RNA pol II subunit RPB1 and hypomethylation of the SL RNA cap, indicating a widespread shutdown of gene expression. Interestingly, blocking trans splicing by silencing the expression of essential splicing factors led to the same defects, strongly indicating that in trypanosomes there is cross-talk between RNA pol II and the spliceosome. Since our preliminary data suggest that CRK9 directly phosphorylates RBP1 and the spliceosomal protein TSR1, and we found CRK9 to be associated with three dual specificity kinases, we hypothesize that CRK9 controls essential mechanistic steps in RNA splicing, RNA pol II-mediated transcription and cell cycle progression either directly or through signaling pathways. Furthermore, CRK9 forms an unusual enzyme complex with a deviant L-type cyclin and a kinetoplastid-specific protein. Thus, we propose to study the cellular function of CRK9 and the architecture of the enzyme complex in detail. This comprises cell-wide surveys of CRK9 silencing defects, functional analyses of CRK9 substrates and their phosphorylation sites, and mapping protein-protein interactions within the trimeric CRK9 complex. Overall, we may unravel and decipher enzyme-mediated pathways that regulate trypanosome gene expression in an unforeseen manner.
Trypanosomatid parasites cause devastating human diseases that affect millions of people worldwide. Drugs to treat these diseases are limited and problematic, and parasite resistance to these drugs is on the rise. As our data indicate, the cyclin-dependent kinase CRK9 forms a unique tripartite protein complex and has essential roles in the coordination of transcription and RNA splicing as well as in the completion of the cell cycle. Thus, analysis of the CRK9 enzyme architecture and of CRK9-controlled substrates, signaling pathways and mechanisms may unravel new chemotherapeutic intervention strategies.
