Chromosome-wide analysis of gene function by RNA interference in the african trypanosome

Trypanosomatids of the order Kinetoplastida are major contributors to global disease and morbidity, and understanding their basic biology coupled with the development of new drug targets represents a critical need. Additionally, trypanosomes are among the more accessible divergent eukaryote experimental systems. The genome of Trypanosoma brucei contains 8,131 predicted open reading frames (ORFs), of which over half have no known homologues beyond the Kinetoplastida and a substantial number of others are poorly defined by in silico analysis. Thus, a major challenge following completion of the T. brucei genome sequence is to obtain functional data for all trypanosome ORFs. As T. brucei is more experimentally tractable than the related Trypanosoma cruzi and Leishmania spp. and shares >75% of their genes, functional analysis of T. brucei has the potential to inform a range of parasite biology. Here, we report methods for systematic mRNA ablation by RNA interference (RNAi) and for phenotypic analysis, together with online data dissemination. This represents the first systematic analysis of gene function in a parasitic organism. In total, 210 genes have been targeted in the bloodstream form parasite, representing an essentially complete phenotypic catalogue of chromosome I together with a validation set. Over 30% of the chromosome I genes generated a phenotype when targeted by RNAi; most commonly, this affected cell growth, viability, and/or cell cycle progression. RNAi against approximately 12% of ORFs was lethal, and an additional 11% had growth defects but retained short-term viability in culture. Although we found no evidence for clustering or a bias towards widely evolutionarily conserved genes within the essential ORF cohort, the putative chromosome I centromere is adjacent to a domain containing genes with no associated phenotype. Involvement of such a large proportion of genes in robust growth in vitro indicates that a high proportion of the expressed trypanosome genome is required for efficient propagation; many of these gene products represent potential drug targets.     

RNA interference mutant induction in vivo demonstrates the essential nature of trypanosome flagellar function during mammalian infection

We demonstrate that trypanosomes compromised in flagellar function are rapidly cleared from infected mice. Analysis of the PFR2 bloodstream RNA interference mutant revealed that defective cell motility occurred prior to cytokinesis failure. This validation provides a paradigm for the flagellum as a target for future assays and interventions against this human pathogen.     

Particle-bound enzymes in the bloodstream form of Trypanosoma brucei.

We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase, isocitrate dehydrogenase, acid phosphatase and part of the hyperoxide dismutase and malate dehydrogenase activities were found to be particle-bound after fractionation of homogenates by differential centrifugation. Part of the ATPase activity was sensitive to oligomycin, an inhibitor of oxidative phosphorylation. This oligomycin-sensitive activity can serve as a specific marker for the mitochondria. More than 80% of the NAD+-linked glycerol-3-phosphate dehydrogenase in T. brucei was found to be particulate and latent. The enzyme could be activated by Triton X-100, by the combined action of sonication and salt, but not by salt alone, and partially by freezing and thawing. We conclude that the NAD+-linked glycerol-3-phosphate dehydrogenase is located inside an organelle.     

A genome‐wide RNA interference screen identifies two novel components of the metazoan secretory pathway

Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome‐wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.     

RNA interference in Escherichia coli cells: the expression of molecules that are complementary to the lon gene mRNA in parallel orientation

To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene.     

Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.

Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.     

The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei

Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase ( Tb ECT) . Gene knockout indicates that Tb ECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei , and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. Tb ECT is therefore genetically validated as a potential drug target against the African trypanosome.     

Methylation interference experiments identify bases that are essential for distinct catalytic functions of a group I ribozyme.

Methylation interference experiments reveal bases involved in three different catalytic functions of the T4-phage derived sunY self-splicing intron. RNA molecules methylated at the N-7 position of the guanine at the cofactor binding site are inactive in cofactor-dependent splicing and 3' splice-site hydrolysis. In contrast, 5' splice-site hydrolysis occurs despite methylation at this position. Specific adenines that have been implicated in docking of the P1 stem to the catalytic core are shown to be important for cofactor-dependent splicing and essential for 5' splice-site hydrolysis. Similarly, methylation of bases in the P9.0 stem, as well as C56 in J5/4, interferes with 3' splice-site hydrolysis and with the splicing reaction. All of the bases identified as important for the overall splicing reaction are also identified as essential for either the 5' or 3' splice-site hydrolysis reactions, and vice versa. It is inferred that the bases implicated in 5' and 3' splice-site hydrolysis are involved in specific interactions of the 5' and 3' splice site, respectively, with the catalytic core.     

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.     

In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif.

Translation initiation factor elF-4B is an RNA-binding protein that promotes the association of the mRNA to the 40S ribosomal subunit. One of its better characterized features is the ability to stimulate the activity of the DEAD box RNA hilicase elF-4A. In addition to an RNA recognition motif (RRM) located near its amino-terimus, elF-4B contains an RNA-binding region in its carboxy-terminal half. The elF-4A helicase stimulatory activity resides in the carboxy-terminal half of elF-4B, and the RRM has little impact on this function.To better understand the role of the elF-4B RRM, it was of interest to identify its specific RNA target sequence. To this end, it vitro RNA selection/amplifications were performed using various portions of elF-4B. These experiments were designed to test the RNA recognition specificity of the two elF-4B regions implicated in RNA binding and to assess the influence of elF-4A on the RNA-binding specificity. The RRM was shown to bind with high affinity to an RNA stem-loop structure with conserved primary sequence elements. Discrete point mutations in an in vitro-selected RNA identified residues critical for RNA binding. Neither the carboxy-terminal RNA-interaction region, nor elF-4A, influenced the structure of the high-affinity RNA ligands selected by elF-4B, and elF-4A by itself did not select any specific RNA target. Previous studies have demonstrated an interaction of elF-4B with ribosomes, and it was suggested that this association is mediated through binding to ribosomal RNA. We show that the RRM of elF-4B interacts directly with 18S rRNA and this interaction is inhibited by an excess of the elF-4B in vitro-selected RNA. ElF-4B could bind simultaneously to two different RNA molecules, supporting a model whereby elF-4B promotes ribosome binding to the 5 untranslated region of a mRNA by bridging it to 18S rRNA.     

An RNA interference screen identifies a novel regulator of target of rapamycin that mediates hypoxia suppression of translation in Drosophila S2 cells

In addition to its central role in energy production, oxygen has pervasive regulatory actions. Hypoxia (oxygen limitation) triggers the shutdown of major cellular processes, including gene expression. We carried out a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells for functions required to down-regulate translation during hypoxia. RNAi knockdown of specific genes allowed induction of a green fluorescent protein (GFP) reporter gene and continued protein synthesis during hypoxia. Among the identified genes, Tsc1 and Tsc2, which together form the tuberose sclerosis complex that negatively regulates target of rapamycin (TOR) kinase, gave an especially strong effect. This finding is consistent with the involvement of TOR in promoting translation. Another gene required for efficient inhibition of protein translation during hypoxia, the protein tyrosine phosphatase 61F (Ptp61F), down-regulates TOR activity under hypoxia. Lack of Ptp61F or Tsc2 improves cell survival under prolonged hypoxia in a TOR-dependent manner. Our results identify Ptp61F as a novel modulator of TOR activity and suggest that its function during hypoxia contributes to the down-regulation of protein synthesis.     

Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes

Plastoglobules are lipoprotein particles inside chloroplasts. Their numbers have been shown to increase during the upregulation of plastid lipid metabolism in response to oxidative stress and during senescence. In this study, we used state-of-the-art high-pressure freezing/freeze-substitution methods combined with electron tomography as well as freeze-etch electron microscopy to characterize the structure and spatial relationship of plastoglobules to thylakoid membranes in developing, mature, and senescing chloroplasts. We demonstrate that plastoglobules are attached to thylakoids through a half-lipid bilayer that surrounds the globule contents and is continuous with the stroma-side leaflet of the thylakoid membrane. During oxidative stress and senescence, plastoglobules form linkage groups that are attached to each other and remain continuous with the thylakoid membrane by extensions of the half-lipid bilayer. Using three-dimensional tomography combined with immunolabeling techniques, we show that the plastoglobules contain the enzyme tocopherol cyclase (VTE1) and that this enzyme extends across the surface monolayer into the interior of the plastoglobules. These findings demonstrate that plastoglobules function as both lipid biosynthesis and storage subcompartments of thylakoid membranes. The permanent structural coupling between plastoglobules and thylakoid membranes suggests that the lipid molecules contained in the plastoglobule cores (carotenoids, plastoquinone, and tocopherol [vitamin E]) are in a dynamic equilibrium with those located in the thylakoid membranes.     

Stem-forming regions that are essential for the amyloidogenesis of prion proteins

Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-β-structure, the initial mechanism in the conversion to a β-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-β-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains ~200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular β-sheet to a solvent through the partial transition of a native structure from an α-helix to a β-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.