Association of a novel pre-ribosomal complex in Trypanosoma brucei

In eukaryotes, 5S rRNA is transcribed in the nucleoplasm and requires the ribosomal protein L5 to deliver it to the nucleolus for ribosome assembly. Trypanosome-specific proteins P34 and P37 form a novel preribosomal complex with the eukaryotic conserved L5-5S rRNA complex in the nucleoplasm. We hypothesize that this novel tri-molecular complex is necessary for stabilizing 5S rRNA in T. brucei and is essential for the survival of the parasite. In vitro quantitative analysis of protein-protein and protein-RNA associations is fundamental to our understanding of this novel complex and the potential for using this complex as a therapeutic target. First, we demonstrated that Fluorescence Energy Resonance Transfer (FRET) can be used to confirm the association between L5 and P34. We then determined that the binding constant for L5 and P34 is 0.60 μM ± 0.03 μM, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we identified the critical regions of L5 and P34 involved in the protein-protein and protein-RNA association. We found that the N-terminal APK rich domain and RNA recognition motif (RRM) of P34 and the L18 domain of L5 are important for both the protein-protein and protein-RNA association. Domains in the two proteins for protein-protein and protein-RNA interaction overlap or are close to each other. We also showed that the T. brucei L5 binds the β arm of 5S rRNA, while P34 binds the Loop A/Stem V of 5S rRNA. Finally, we demonstrated that 5S rRNA is able to enhance the association between L5 and P34 using FRET. Both the Loop A/Stem V and β arm of 5S rRNA can separately enhance the protein-protein association, but the enhancement is not increased when they are added together. This suggests that 5S rRNA binding may cause conformational changes in L5, P34 and 5S rRNA, thus bridging and enhancing binding between the protein partners of this novel complex. These results have identified critical features of both proteins and RNA that are part of this trypanosome-specific preribosomal complex and provide us with the framework for the discovery of ways to disrupt this essential complex.