Association of a Novel Preribosomal Complex in Trypanosoma brucei Determined by Fluorescence Resonance Energy Transfer

Abstract

We have previously reported that the trypanosome-specific proteins P34 and P37 form a unique preribosomal complex with ribosomal protein L5 and 5S rRNA in the nucleoplasm. We hypothesize that this novel trimolecular complex is necessary for stabilizing 5S rRNA in Trypanosoma brucei and is essential for the survival of the parasite. In vitro quantitative analysis of the association between the proteins L5 and P34 is fundamental to our understanding of this novel complex and thus our ability to exploit its unique characteristics. Here we used in vitro fluorescence resonance energy transfer (FRET) to analyze the association between L5 and P34. First, we demonstrated that 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 ؎ 0.03 M, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we used FRET to identify the critical regions of L5 and P34 involved in the protein-protein 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 the association of the two proteins with each other. These results provide us with the framework for the discovery of ways to disrupt this essential complex.A frican trypanosomes are responsible for trypanosomiasis in humans and nagana in domestic animals. Trypanosoma brucei has a complicated life cycle that requires adaptation to life within a mammalian host and an insect vector, the tsetse fly (1). Throughout this life cycle, RNA binding proteins (RBPs) play the central roles in many adaptive processes, including regulation of gene expression and protein synthesis (2, 3), making them particularly important in this organism.RBPs play essential roles in the many aspects of ribosomal biogenesis. Ribosomal biogenesis in eukaryotes is an essential and conserved process that requires the processing and assembly of four rRNAs (18, 28, 5.8, and 5S rRNAs) with over 80 ribosomal proteins. Much of this process occurs in the nucleolus (4-6). However, unlike the other three rRNAs, 5S rRNA is independently transcribed by RNA polymerase III within the nucleoplasm and must be transported into the nucleolus by the L5 ribosomal protein (7,8). L5 is the only known eukaryotic ribosomal protein that forms a preribosomal L5-5S rRNA ribonucleoprotein (RNP) complex to stabilize and facilitate the trafficking of 5S rRNA to the nucleolus (7, 9, 10). Mutations in critical residues within L5 lead to loss of 5S rRNA and ribosomal biogenesis defects, indicating that it is essential to cell viability. T. brucei L5 is comprised of a predicted RanBP1_WASP domain in its N-terminal region, an L18 domain in the central region, and a C-terminal domain that possesses residues critical for 5S rRNA binding.Our laboratory has previously identified two trypanosomespecific RBPs, P34 and P37, which are essential for the viability of T. brucei (11). P34 and P37 are highly similar, the major difference between them being the pre...

Defining the RNA-Protein Interactions in the Trypanosome Preribosomal Complex

“…L5 (GenBank accession no. XM_822569), cerulean-L5, enhanced yellow fluorescent protein (eYFP)-P34, the N and C termini and L18 domain of L5, and the N and C termini and RRM domain of P34 were cloned into pTrcHis-TOPO (Invitrogen) for expression as polyhistidine fusion proteins in TOP10 One Shot cells (Invitrogen), as described previously (16). All of the recombinant protein expression plasmids were confirmed by DNA sequence analysis.…”

“…showed that the N-terminal APK-rich domain and the RRM domain of P34 and the L18 domain of L5 are important for the association between P34 and L5 (16). Immune capture assays also showed that 5S rRNA enhances the protein-protein association (11).…”

“…We have analyzed domains of L5 and P34 for proteinprotein interactions and the regions of 5S rRNA for protein-RNA interactions (10,16). It has been shown that the N-terminal APKrich domain and the RRM domain of P34 and the L18 domain of L5 are important for the association of the two proteins with each other (16). We have shown also that P34 and P37 bind 5S rRNA through a high-affinity interaction with the loop A/stem V region (10).…”

“…The T. brucei L5 is composed of a predicted RanBP1_WASP domain in the N-terminal region, an L18 domain in the central region, and a C-terminal domain which contains an ␣ helix with a less-dense concentration of positive charge than in other eukaryotic L5 proteins (11). We have analyzed domains of L5 and P34 for proteinprotein interactions and the regions of 5S rRNA for protein-RNA interactions (10,16). It has been shown that the N-terminal APKrich domain and the RRM domain of P34 and the L18 domain of L5 are important for the association of the two proteins with each other (16).…”

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

“…Immunoprecipitations were performed as previously described (24). Protein A-labeled magnetic beads (Dynabeads, Life Technologies) were blocked in phosphate-buffered saline (PBS)-bovine serum albumin and incubated with anti-P34/P37 or anti-L5 antibody for 1 h at 4°C.…”