Conserved and trypanosome specific factors in ribosomal biogenesis of Trypanosoma brucei and Trypanosoma cruzi

Trypanosoma brucei spp are single celled parasites and are the causative agents of human African trypanosomiasis (HAT) as well as nagana in animals. Together with Trypanosoma cruzi and Leishmania spp, they are part of a group of divergent early branching eukaryotic pathogens known as the TriTryps. There is an urgent need for effective new drugs against these organisms that cause the important neglected tropical diseases HAT, Chagas disease, and leishmaniasis. A major part of the effort to develop new drugs is discovering new drug targets. Our group has previously identified two essential proteins in T. brucei, P34 and P37, which have no known homologs in organisms outside of the TriTryps. These proteins form a novel trimolecular preribosomal complex together with 5S rRNA and L5 ribosomal protein. P34 and P37 are essential for 5S rRNA stability and ribosomal biogenesis and the effect of their depletion in T. brucei is similar to the effect of L5 depletion in other organisms. Although T. brucei L5 can bind 5S rRNA in vitro, the affinity of this interaction is five fold lower than the value for typical eukaryotic L5. This observation led us to postulate that T. brucei L5 function might be different from that in other organisms. We used RNA interference to investigate L5 function in T. brucei. Using this approach we showed that L5 is an essential protein in T. brucei, despite the presence of other 5S rRNA binding proteins, P34 and P37. Loss of L5 is accompanied by a dramatic and specific drop in the levels of both rRNA and protein components of the large (60S) ribosomal subunit and in stable assembly of that subunit. Further, we found that loss of L5 impacted the maturation and processing of ribosomal RNAs with precursor rRNAs accumulating. To our surprise, some of these precursors are found in association with cytoplasmic ribosomes and polysomes. This constitutes a novel finding and indicates a loss of the quality control mechanisms that normally exclude immature rRNA species from reaching cytoplasmic ribosomes. P34 and P37 levels are increased in L5 depleted trypanosomes particularly in association with ribosomes. This suggests that a compensatory mechanism exists whereby T. brucei upregulates levels of one set of 5S rRNA-binding proteins, P34/P37, in response to depletion of another 5S rRNA-binding factor, L5.;In order to ascertain the extent of the conservation of P34/P37 functions in other Tritryps, we characterized the T. cruzi homolog(s) of P34/P37. We first determined that there is a single homolog, TcP37, which shares a number of features with TbP34 and TbP37. These include the presence of an N-terminal APK rich domain and two canonical RNA recognition motifs (RRMs) which typically bind RNAs, but can also bind proteins. However, TcP37 lacks the C-terminal domain present in TbP34/TbP37. This C-terminal region contains nuclear localization signals as well as nucleolar localization signals. Despite the absence of this region, TcP37 localizes to the nucleus (including nucleolus) as well as the cytoplasm. Like its T. brucei homologs, TcP37 is an efficient 5S rRNA binding protein in vitro. This binding is specific since polyadenylated RNAs do not associate with TcP37. In addition, we demonstrated that TcP37 also associates both in vivo and in vitro with the L5 ribosomal protein and is part of a complex containing both 5S rRNA and L5. As was previously reported for P34 in T.brucei, TcP37 can bind L5 in the absence of any other factors, but unlike TbP37 the addition of 5S rRNA strongly enhances the protein-protein association in vitro and the in vivo association between TcP37 and L5 can be largely disrupted by RNAses. These results suggest that loss of the C-terminal domain impacts the protein-protein association which may be augmented by 5S rRNA.;In summary, we show that the 5S rRNA pathway and ribosomal biogenesis in trypanosomes possess conserved and non-conserved essential factors. The biochemical features of the trypanosome-specific factors TcP37 and TbP34/P37 are well conserved. Our work sets the foundation to explore strategies to disrupt the essential interactions between these components and the conserved partners L5 and 5S rRNA across several species of pathogenic trypanosomes.