Analysis of the Unique Binding Interactions within the 5S Ribonucleoprotein Particle (5S RNP) in Trypanosoma brucei.

摘要

Ribosomes are the cellular machines responsible for protein synthesis. Several unique features about ribosome biogenesis have been characterized in Trypanosoma brucei, the causative agent of African trypanosomiasis in humans and animals. One unique feature identified by our laboratory, is the involvement of two essential trypanosome-specific RNA binding proteins, P34 and P37, in the maturation of the 60S ribosomal subunit. Our laboratory has shown that knockdown of these essential proteins leads to a defect in ribosome formation and an overall decrease in protein synthesis. We have also shown that together with 5S ribosomal RNA (rRNA) and ribosomal protein L5, P34 and P37 form the T. brucei 5S ribonucleoprotein particle (5S RNP).;In this work we have analyzed the in vitro protein-protein and protein-RNA interactions of P34 with 5S rRNA and with L5 and shown that P34 mediates both interactions via its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine how the domains mediate binding and we also mutated specific residues in P34 to disrupt binding with 5S rRNA and L5 protein. We determined that the RRM1 domain of P34 mediates most of the interaction with 5S rRNA and that the N-terminus and RRM1 domains combined mediate the interaction with L5 protein. We determined that the RRM domains of P34 contain ribonucleoprotein (RNP) 1 and 2 consensus sequences that, in canonical RRM domains, have been shown to mediate sequence specific binding with their RNA target. In P34 these sequences are partially conserved. However, the aromatic amino acids implicated in mediating binding with RNA are conserved. Therefore, we mutated these residues to alanine to disrupt the interaction between P34 and 5S rRNA. Surprisingly, mutating these sites did not disrupt but rather increased the binding affinity of 5S rRNA for P34. This suggests that these residues in P34 are not involved in mediating direct binding to 5S rRNA. We identified four tandem arginine residues in RRM1 of P34 that when mutated to alanine led to a significant decrease in binding with L5. Collectively these mutational studies led us to the model that the binding sites for both 5S rRNA and L5 to P34 protein are near each other in RRM1 and that specific residues in the RRM1 domain of P34 could regulate binding of 5S rRNA and L5 to P34.;In eukaryotes, the 5S RNP is composed of additional members; ribosomal protein L11 and assembly factors Rpf2 and Rrs1. These additional members have not been characterized in T. brucei. We generated tagged cell lines in order to isolate proteins that associate with L5, P34, and P37 proteins. Using mass spectrometry analysis to identify the associating proteins from the pull down, we identified the homolog of the assembly factor Rpf2 protein. We determined that TbRpf2 makes conserved interactions with 5S rRNA and L5 protein both in vivo and in vitro. Interestingly, we also showed that TbRpf2 interacts with P34 and P37 in vivo and with P34 in vitro. We used RNA interference to show that TbRpf2 is an essential protein and that loss of this protein led to an accumulation of the 40S subunit and a decrease in 80S monosome formation. These results suggest that the role of Rpf2 as described in S. cerevisiae, is conserved in T. brucei.;Overall, we have characterized several unique features of the interactions among members of the T. brucei 5S RNP. The studies of the domains and residues of P34 that mediate binding with 5S rRNA and with L5 protein has allowed for a better understanding of how the RRM domains of P34 are able to mediate both protein-protein and protein-RNA interactions. Characterization of an additional member of the 5S RNP has shown that TbRpf2 is an essential protein that not only associates with conserved components but also with trypanosome-specific components of this complex. Collectively, our studies have provided a better understanding of the unique features of the T. brucei 5S RNP complex that may be druggable.;..