Functional characterization of two RNA binding proteins, P34 and P37, from Trypanosoma brucei
Prohaska, Kimberly M.
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Our laboratory has previously identified and purified two nearly identical RNA binding proteins, P34 and P37, from Trypanosoma brucei. The only differences between these two proteins are an 18 amino acid insert in the N-terminus of P37 and four single amino acid differences with respect to P34. Initial experiments were performed to elucidate molecular binding partners of P34 and P37, which demonstrated interactions with a family of nucleolar phosphoproteins, NOPP44/46, ribosomal protein L5, and 5S rRNA. To further characterize the association of P34 and P37 with NOPP44/46, a P34/P37 RNAi cell line was utilized, in which expression of both proteins was simultaneously knocked down. In the absence of these proteins, NOPP44/46 nuclear protein levels increased approximately 12-fold, suggesting a role for P34 and P37 in regulating NOPP44/46 expression. It was then demonstrated that P34 and P37 do not regulate NOPP44/46 at the level of transcript abundance or stability, or at the level of total cellular protein. Results from immune capture experiments showed that P34 and P37 associate with exportin 1, a nuclear export factor and, that they mediate an association between this protein and NOPP44/46, thereby regulating their cellular localization. Since NOPP44/46, L5, and 5S rRNA are involved at some stage during biogenesis of the 60S ribosomal subunit and each associates with P34 and P37, it was hypothesized that P34 and P37 are also involved in this pathway. Immune capture experiments specific for ribosomal proteins which enter the 60S biogenesis pathway at different points were performed to determine when and where P34 and P37 come into the pathway. The results from these experiments showed that P34 and P37 enter into the pathway at the early 90S pre-ribosomal particle in the nucleolus and that they remain associated subsequent to nuclear export and subunit joining. Interestingly, experiments performed using the P34/P37 RNAi cells demonstrated that in the absence of P34 and P37, the 60S subunit no longer interacts with XpoI or Nmd3, components of the nuclear complex required for the export of the yeast 60S subunit. These results support an essential role for P34 and P37 in the nuclear export of the 60S subunit in trypanosomes. Together, the results presented in this thesis demonstrate the potential for multi-functional roles for P34 and P37 in the ribosomal biogenesis pathway. These studies lay the groundwork for further experiments aimed at more specifically determining the function(s) of P34 and P37 in ribosomal biogenesis.