TbMP44 is essential for RNA editing and structural integrity of the editosome in Trypanosoma brucei

RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation of TbMP44 gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.     

The RNA editing process in Trypanosoma brucei

Twelve mitochondrial mRNAs are edited in Trypanosoma brucei, nine extensively, by addition and removal of uridines. The accumulation of the edited RNAs is regulated during the life cycle. Hundreds of different gRNAs, encoded three or four per minicircle, specify the editing and minicircle content accounts for variation in editing among species and in mutants. The current understanding of the process of gRNA utilization, the editing mechanism and the editing machinery is discussed.     

Structural flexibility in Trypanosoma brucei enolase revealed by X-ray crystallography and molecular dynamics

Enolase is a validated drug target in Trypanosoma brucei. To better characterize its properties and guide drug design efforts, we have determined six new crystal structures of the enzyme, in various ligation states and conformations, and have carried out complementary molecular dynamics simulations. The results show a striking structural diversity of loops near the catalytic site, for which variation can be interpreted as distinct modes of conformational variability that are explored during the molecular dynamics simulations. Our results show that sulfate may, unexpectedly, induce full closure of catalytic site loops whereas, conversely, binding of inhibitor phosphonoacetohydroxamate may leave open a tunnel from the catalytic site to protein surface offering possibilities for drug development. We also present the first complex of enolase with a novel inhibitor 2-fluoro-2-phosphonoacetohydroxamate. The molecular dynamics results further encourage efforts to design irreversible species-specific inhibitors: they reveal that a parasite enzyme-specific lysine may approach the catalytic site more closely than crystal structures suggest and also cast light on the issue of accessibility of parasite enzyme-specific cysteines to chemically modifying reagents. One of the new sulfate structures contains a novel metal-binding site IV within the catalytic site cleft.     

TbRGG2, an essential RNA editing accessory factor in two Trypanosoma brucei life cycle stages

In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 down-regulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.     

Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.     

Naphthalene-based RNA editing inhibitor blocks RNA editing activities and editosome assembly in Trypanosoma brucei

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components.     

RNA editing in Trypanosoma brucei requires three different editosomes

Trypanosoma brucei has three distinct ~20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ~20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

TbMP81 is required for RNA editing in Trypanosoma brucei

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.     

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.     

Four related proteins of the Trypanosoma brucei RNA editing complex

RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact.     

Functional conformations of the L11-ribosomal RNA complex revealed by correlative analysis of cryo-EM and molecular dynamics simulations

The interaction between the GTPase-associated center (GAC) and the aminoacyl-tRNA.EF-Tu.GTP ternary complex is of crucial importance in the dynamic process of decoding and tRNA accommodation. The GAC includes protein L11 and helices 43-44 of 23S rRNA (referred to as L11-rRNA complex). In this study, a method of fitting based on a systematic comparison between cryo-electron microscopy (cryo-EM) density maps and structures obtained by molecular dynamics simulations has been developed. This method has led to the finding of atomic models of the GAC that fit the EM maps with much improved cross-correlation coefficients compared with the fitting of the X-ray structure. Two types of conformations of the L11-rRNA complex, produced by the simulations, match the cryo-EM maps representing the states either bound or unbound to the aa-tRNA.EF-Tu.GTP ternary complex. In the bound state, the N-terminal domain of L11 is extended from its position in the crystal structure, and the base of nucleotide A1067 in the 23S ribosomal RNA is flipped out. This position of the base allows the RNA to reach the elbow region of the aminoacyl-tRNA when the latter is bound in the A/T site. In the unbound state, the N-terminal domain of L11 is rotated only slightly, and A1067 of the RNA is flipped back into the less-solvent-exposed position, as in the crystal structure. By matching our experimental cryo-EM maps with much improved cross-correlation coefficients compared to the crystal structure, these two conformations prove to be strong candidates of the two functional states.     

RNA sequence and base pairing effects on insertion editing in Trypanosoma brucei

RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.     

Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex

The Trypanosoma brucei editosome catalyzes the maturation of mitochondrial mRNAs through the insertion and deletion of uridylates and contains at least 16 stably associated proteins. We examined physical and functional associations among these proteins using three different approaches: purification of complexes via tagged editing ligases TbREL1 and TbREL2, comprehensive yeast two-hybrid analysis, and coimmunoprecipitation of recombinant proteins. A purified TbREL1 subcomplex catalyzed precleaved deletion editing in vitro, while a purified TbREL2 subcomplex catalyzed precleaved insertion editing in vitro. The TbREL1 subcomplex contained three to four proteins, including a putative exonuclease, and appeared to be coordinated by the zinc finger protein TbMP63. The TbREL2 subcomplex had a different composition, contained the TbMP57 terminal uridylyl transferase, and appeared to be coordinated by the TbMP81 zinc finger protein. This study provides insight into the molecular architecture of the editosome and supports the existence of separate subcomplexes for deletion and insertion editing.     

The adenosine analog tubercidin inhibits glycolysis in Trypanosoma brucei as revealed by an RNA interference library

We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAi-based genomic library, we used 5 microm tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidin-resistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC50 of 7.5 microm. We conclude that 5 microm tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.     

Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase.

RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation.