Previous work from this laboratory has focused on the role of two trypanosome-specific RNA binding proteins, P34/P37, in the largely conserved ribosomal biogenesis pathway. The work proposed here will expand our studies into the larger question of the unique features of trypanosome ribosomal biogenesis. Recent cryo-EM studies from our collaborator, Dr. Frank (Columbia University), have shown that the T. brucei ribosome possesses a number of unique features including large expansion segments in the ribosomal RNAs that suggest sites for docking of trypanosome-specific proteins or trypanosome-specific extensions of the otherwise conserved ribosomal proteins. However, their studies were limited in that they focused on the cytoplasmic ribosome and used relatively harsh conditions to prepare them. So although some interesting features of the ribosome "core" proteins were found, they were unable to identify any unique ribosomal proteins. A number of laboratories have characterized non-"core" RNA binding proteins that are essential to ribosomal biogenesis in trypanosomes. This suggests that these trypanosome-specific proteins (both those already identified and those yet to be discovered) are more peripheral or have a more transient association and yet are essential to ribosomal maturation. The studies described here will identify trypanosome-specific proteins that interact with the ribosome during the biogenesis pathway and, in particular, interact with these unique expansion segments. We will also examine the unique features of the exportin 1 (XpoI) export complex responsible for 60S (and likely 40S) export from the nucleus to the cytoplasm and determine whether other export receptor systems are also used. Our hypothesis is that the function of these proteins will provide valid targets for intervention in the ribosomal assembly in T. brucei.
The specific aims of the project address the following questions. 1. What unique proteins interact with the ribosome? 2. Which proteins interact with the expansion segments and are they conserved or unique to trypanosomes? 3. How do the 60S and 40S complexes interact with the nuclear export complex in trypanosomes? Treatment of diseases caused by trypanosomes is severely limited by the lack of new drugs as well as resistance, toxicity, and issues with cost and administration for the few drugs currently available. Ribosomal structure and function have been identified as targets for antibiotics in other systems. By determining additional unique features of kinetoplastid ribosomal structure and biogenesis we will identify new targets for chemotherapeutic intervention that are desperately needed for these parasites.
The protozoan parasites, the Kinetoplastids, include the organisms Trypanosoma brucei, Trypanosoma cruzi and Leishmania, which cause serious diseases in much of the world (African sleeping sickness, Chagas'disease and leishmaniasis, respectively). This study will examine the unique trypanosomal features of the ribosomal biogenesis pathway to identify new chemotherapeutic targets for these parasites.