Analysis of a novel pre-ribosomal complex in Trypanosoma brucei
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Trypanosoma brucei is the causative agent of human African trypanosomiasis. It belongs to the kinetoplastids, a group of early-branching eukaryotes with many unique biochemical and genetic characteristics. Our laboratory had previously identified two abundant RNA binding proteins specific to the parasitic trypanosomatids T. brucei, T. cruzi and Leishmania. These proteins, P34 and P37, are essential in T. brucei. Previous data indicated that P34 and P37 associate with 5S rRNA in the nucleus. In silico analysis of the T. brucei 5S rRNA yielded two different structures with similar energetic parameters. Both structures are organized in five stems and five loops. One of them has a typically eukaryotic Loop C, while the other has an unusually reduced Loop C. RNase H digestion patterns were consistent with a form in which loop C is exposed and available for inter-molecular interactions. Studies using recombinant proteins demonstrated that both P34 and P37 can bind 5S rRNA with high specifity and affinity in the absence of additional cellular factors. Different approaches were taken to investigate which domains on the 5S rRNA are involved in the interaction with P34 and P37. We conclude that the Loop A / Stem V region in 5S rRNA is an important interaction site for the association with P34 and P37. However, upon binding, both P34 and P37 protect a large area of the molecule which extends to Loop C. In other eukaryotes, the Loop A / Stem V region is involved in an interaction with TFIIIA, a factor that has not been identified in the T. brucei databases. Loop C mediates a high affinity, conserved interaction with ribosomal protein L5. This association serves to stabilize the nascent 5S rRNA, and transport it to the nucleolus. The L5-5S rRNA RNP is the only characterized pre-ribosomal particle in eukaryotes to date. In silico studies of the T. brucei L5 protein reveal deviation from the eukaryotic consensus at potentially key positions implicated in binding to 5S rRNA. In vivo studies demonstrate that the T. brucei L5 protein binds a lower percentage of total 5S rRNA than previously characterized L5 proteins. In addition, in vitro studies with recombinant proteins show that the affinity of T. brucei L5 for 5S rRNA is 20-fold lower than that of the Xenopus laevis L5 protein. A mutation in the T. brucei C-terminus domain of L5 that restores a positive charge within an alpha helix to consensus increases binding to 5S rRNA. In T. brucei cells lacking P34 and P37, 5S rRNA is destabilized and ribosomal biogenesis is impaired. A similar phenotype is observed in yeast cells depleted of L5. We hypothesize that in trypanosomes, L5 is less efficient at binding 5S rRNA, and that P34 and P37 have been recruited to compensate L5 function.