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.

The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F 1 -ATPase ␣ and ␤ subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the ␤ subunit decreased the mRNA and protein levels for the ␤ subunit, as well as the mRNA and protein levels of the ␣ subunit. Similarly, RNAi of ␣ subunit decreased the ␣ subunit transcript and protein levels, as well as the ␤-subunit transcript and protein levels. In contrast, ␣ and ␤ RNAi knockdown resulted in a 60% increase in the F 0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F 0 -32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for ␣ or ␤ subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of ␣ and ␤ RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.The mitochondrial ATP synthase couples the electrochemical proton gradient to the synthesis or hydrolysis of ATP (5,11,26,39). The ATP synthase is composed of the soluble F 1 moiety, which contains the catalytic sites and the membranebound F 0 moiety, which is involved in proton translocation. The F 1 moiety of the mitochondrial ATP synthase is highly conserved and is composed of five subunits present in a stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 ε 1 . The F 0 moiety in Escherichia coli is composed of three subunits, a 1 b 2 c 10-14 , but in eukaryotes its subunit composition increases in complexity to include up to eight additional subunit types (5,22,26). The Trypanosoma brucei mitochondrial ATP synthase has been isolated and characterized. The molecular composition of the enzyme complex is similar to that of other eukaryotic mitochondrial ATP synthases (43). Both functional assays and analysis of protein levels indicate that the complex is developmentally regulated through the life cycle of the organism (7,41,42).A striking feature of T. brucei is its ability to adapt to diverse environments encountered through the stages of its life cycle (21, 34). In the tsetse fly, the mitochondrion of the procyclic trypanosomes is fully developed with many cristae, a complete respiratory chain, Krebs cycle enzymes, and abundant levels of mitochondrial ATP synthase. In contrast, the sparse and tubular mitochondrion of the early (slender) mammalian blood...

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 ...

Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated H1MaI, comprises lipid and two membrane proteins, ll-AM and II-BMaI. The proteins are encoded by ptsM, located at "40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for X DNA penetration of the cytoplasmic membrane. Earlier studies suggested that bothpel function and ll-Bm are encoded by the same gene, while a different gene (also in ptsM) encodes II-AM". In the present studies, a ptsM done, pCS13, was isolated from an E. colU Hindml gene bank in pBR322 and restored both mannose fermentation and pelt function to ptsM mutants defective in II-BMaI. Subclones of pCS13 show that (i) two distinct genes, manY and manZ, encode thepel function and the II-BMa`protein, respectively; (ii) each gene may have its own promoter; (iii) whereas the protein encoded by manY (Pel) alone seems sufficient for X sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the HIMan complex. The efficiency of the complex may depend on the ratio of Pel to 11Man.The phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the translocation of its sugar substrates across the bacterial membrane concomitant with their phosphorylation (1-3). The PTS consists of two cytoplasmic phosphorylated carrier proteins, enzyme I and HPr (which are not sugar specific) and a number of sugar-specific membrane-associated proteins, called enzyme II complexes. Phosphoryl group transfer occurs in the following sequence: phosphoenolpyruvate -* enzyme I --HPr --enzyme II sugar.Two enzyme II complexes catalyze glucose translocation across the Escherichia coli membrane (4, 5). One, designated IIGlc, is specific for glucose and methyl glucosides. The second complex, IIMan, the subject of this report, comprises two membrane-associated proteins II-AMan and II-BMan, and phosphorylates glucose, mannose, and their analogues, such as 2-deoxyglucose (4-7).The concept of two proteins in the IIMan complex was based on early biochemical data (7). By contrast, genetic mapping suggested only a single locus for ptsM (8), at =40 minutes on the E. coli map. Another mutation in this region, called pel, resulted in resistance to penetration of the bacterial inner membrane by X DNA (9, 10). pel appeared to be closely related to ptsM since strains bearing mutations in pel were unable to ferment mannose (although only 30% of the strains unable to ferment mannose were insensitive to X phage).Recent work in this and other laboratories (refs. 11 and 12; P. Saris and E. T. Palva, personal communication) has shown thatptsM contains two structural genes-one encodes II-AMan (manX), and the other encodes II-BMan (manZ). Transcription occurs from manX through manZ with a str...

We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.

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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...