The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function.

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis.

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-aminoacid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a highermolecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.Ribosomes are essential in all organisms, and their assembly is highly conserved and coordinated. Over 100 accessory proteins are necessary in order for proper processing of ribosomal RNAs and ribosome assembly to occur (8). Ribosomal proteins must be imported from the cytoplasm. The 45S precursor rRNA must be processed to yield 5.8S, small-subunit (SSU) (18S), and large-subunit (LSU) (28S) rRNAs. 5S rRNA, which is independently transcribed within the nucleoplasm, must be imported into the nucleolus by the L5 ribosomal protein for ribosome assembly to occur (16). Ribosomal subunits are subsequently exported to the cytoplasm, where the pre-40S ribosomal subunit undergoes its final processing step (29). In eukaryotes, RNA binding proteins mediate a variety of cellular activities, including mRNA maturation, trafficking, stability, and translational control of mRNA as well as having roles in ribosomal biogenesis (14).The parasite Trypanosoma brucei and its subspecies cause human sleeping sickness (T. brucei gambiense and T. brucei rhodesiense) and nagana in livestock (T. brucei brucei) (31). These organisms continue to pose a serious threat to human health and to cause devastating economic losses (1). Little is currently known about RNA binding proteins and small nucleolar RNAs that are involved in rRNA processing and posttranscriptional modifications in T. brucei. Two proteins with homology to 5S rRNA binding proteins in higher eukaryotes, the La autoantigen and the ribosomal L5 protein, have been identified in T. brucei (19, 34) A family of nucleolar phosphoproteins termed NOPP44/46 proteins have also been identified in this organism and implicated in large ...

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/ 28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention. Biosynthesis of proteins is an essential process in all living cells. Ribosomes, large ribonucleoprotein complexes, are responsible for this process across all domains of life. They are composed of two subunits. In eukaryotes, the 40S small subunit (SSU) contains a single 18S rRNA and more than 30 proteins, while the 60S large subunit (LSU) consists of three rRNA molecules (25/28S, 5.8S, and 5S rRNAs) as well as more than 40 proteins (1). While the rRNAs perform the catalytic function of protein synthesis, the protein components provide the structural scaffold of the ribosome and contribute to shaping interaction sites with accessory factors (2-4). In addition to ribosomal proteins, more than 170 accessory proteins are involved in the ribosomal biogenesis pathway. These proteins participate in the maturation, trimming, and modification of the rRNAs, the transport of the precursors across different cellular compartments, and the assembly of a translation-competent ribosome (5). The pathway of ribosome biogenesis is evolutionarily conserved throughout the eukaryotic phylogenetic tree. Early stages of ribosomal biogenesis take place in the nucleolus, where all of the rRNAs except 5S rRNA are transcribed as a single large primary transcript by RNA polymerase I and are processed into 5.8S, 18S, and 25/28S rRNAs (6). In eukaryotes, 5S rRNA is a small and essential rRNA of 120 nucleotides whose function in...

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC.

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Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

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...