Characterization of the protein-protein interactions and RNA binding properties of p34 and p37 from Trypanosoma brucei

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, from Trypanosoma brucei. These proteins have been shown to bind 5S rRNA and a family of nucleolar phosphoproteins, NOPP44/46. In the studies presented here, we have employed RNA interference studies in order to gain further insight into the protein-protein and protein-RNA interactions of p34 and p37 in T. brucei.; Loss of p34 and p37 results in disruption of a higher molecular weight complex containing 5S rRNA, as well as a dramatic decrease in 5S rRNA levels suggesting that these proteins serve to stabilize 5S rRNA. No change in ribosome assembly was found although a significant decrease in overall protein synthesis occurred within p34/p37 RNAi cells. We next evaluated the relationship of p34 and p37 with other conserved 5S rRNA binding proteins. We showed that p34 and p37 do not stably interact with the La protein although this protein is able to form a higher molecular weight complex(es) in the absence of p34 and p37. La protein levels exhibited a modest increase in p34/p37 RNAi cells. Loss of p34 and p37 and subsequent loss of 5S rRNA does not effect the participation of the L5 ribosomal protein in complex formation or L5 protein levels. We found that p34 and p37 bind to the L5 ribosomal protein. The amount of 5S rRNA bound to p34 and p37 is similar to the amount bound by L5.; The loss of p34 and p37 in our RNAi cell lines also led to disruption of a higher molecular weight complex containing the NOPP44/46 proteins as well as a dramatic 12-fold increase in NOPP protein levels within the nucleus. No changes occurred in either NOPP44/46 mRNA steady state levels or stability indicating that p34 and p37 do not affect NOPP expression post-transcriptionally. Surprisingly, we found no alterations in NOPP protein levels in total cell extracts from p34/p37 RNAi cells, in sharp contrast to the increase in NOPP nuclear extracts from these same cells. These results have led us to propose that p34 and p37 function in the regulation of NOPP44/46 intracellular localization.