The RNA chaperone activity of the Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs.
Ontology highlight
ABSTRACT:
SUBMITTER: Leeder WM
PROVIDER: S-EPMC4726059 | biostudies-literature | 2016 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Sequence-deficient mitochondrial pre-mRNAs in African trypanosomes are substrates of a U-nucleotide-specific RNA editing reaction to generate translation-competent mRNAs. The reaction is catalyzed by a macromolecular protein complex termed the editosome. Editosomes execute RNA-chaperone activity to overcome the highly folded nature of pre-edited substrate mRNAs. The molecular basis for this activity is unknown. Here we test five of the OB-fold proteins of the Trypanosoma brucei editosome as candidates. We demonstrate that all proteins execute RNA-chaperone activity albeit to different degrees. We further show that the activities correlate to the surface areas of the proteins and we map the protein-induced RNA-structure changes using SHAPE-chemical probing. To provide a structural context for our findings we calculate a coarse-grained model of the editosome. The model has a shell-like structure: Structurally well-defined protein domains are separated from an outer shell of intrinsically disordered protein domains, which suggests a surface-driven mechanism for the chaperone activity.
| S-EPMC6212840 | biostudies-literature
Project description:The assembly of high molecular mass ribonucleoprotein complexes typically relies on the binary interaction of defined RNA sequences or precisely folded RNA motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome, we identified multiple similar but non-identical RNA motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.5477 (λ2). We establish that synthetic 4V(3E)-RNAs are sufficient to compete for the editosomal pre-mRNA binding site and that they inhibit RNA editing in vitro. Furthermore, we demonstrate that only two topological indices are necessary to predict the binding of any RNA motif to the editosome with a high level of confidence. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA folding space by recognizing a fuzzy continuum of RNA folds that fit a consensus graph descriptor.
| S-EPMC9178004 | biostudies-literature
Project description: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.
| S-EPMC3077619 | biostudies-literature
Project description:The 20S editosome, a multiprotein complex, catalyzes the editing of most mitochondrial mRNAs in trypanosomatids by uridylate insertion and deletion. RNAi mediated inactivation of expression of KREPA4 (previously TbMP24), a component of the 20S editosome, in procyclic form Trypanosoma brucei resulted in inhibition of cell growth, loss of RNA editing, and disappearance of 20S editosomes. Levels of MRP1 and REAP-1 proteins, which may have roles in editing but are not editosome components, were unaffected. Tagged KREPA4 protein is incorporated into 20S editosomes in vivo with no preference for either insertion or deletion subcomplexes. Consistent with its S1-like motif, recombinant KREPA4 protein binds synthetic gRNA with a preference for the 3' oligo (U) tail. These data suggest that KREPA4 is an RNA binding protein that may be specific for the gRNA Utail and also is important for 20S editosome stability.
| S-EPMC1440894 | biostudies-literature
Project description:Multiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ~20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5' OH on the 3' product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei.
| S-EPMC3416509 | biostudies-literature
Project description:RNA editing is an essential post-transcriptional process that creates functional mitochondrial mRNAs in Kinetoplastids. Multiprotein editosomes catalyze pre-mRNA cleavage, uridine (U) insertion or deletion, and ligation as specified by guide RNAs. Three functionally and compositionally distinct editosomes differ by the mutually exclusive presence of the KREN1, KREN2 or KREN3 endonuclease and their associated partner proteins. Because endonuclease cleavage is a likely point of regulation for RNA editing, we elucidated endonuclease specificity in vivo. We used a mutant gamma ATP synthase allele (MGA) to circumvent the normal essentiality of the editing endonucleases, and created cell lines in which both alleles of one, two or all three of the endonucleases were deleted. Cells lacking multiple endonucleases had altered editosome sedimentation on glycerol gradients and substantial defects in overall editing. Deep sequencing analysis of RNAs from such cells revealed clear discrimination by editosomes between sites of deletion versus insertion editing and preferential but overlapping specificity for sites of insertion editing. Thus, endonuclease specificities in vivo are distinct but with some functional overlap. The overlapping specificities likely accommodate the more numerous sites of insertion versus deletion editing as editosomes collaborate to accurately edit thousands of distinct editing sites in vivo.
| S-EPMC5416837 | biostudies-literature
Project description:Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3'-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages.
| S-EPMC4599000 | biostudies-literature
Project description:The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds.
| S-EPMC4756849 | biostudies-literature
Project description:Expression of mitochondrial genomes in Kinetoplastida protists requires massive uracil insertion/deletion mRNA editing. The cascade of editing reactions is accomplished by a multiprotein complex, the 20S editosome, and is directed by trans-acting guide RNAs. Two distinct RNA terminal uridylyl transferases (TUTases), RNA Editing TUTase 1 (RET1) and RNA Editing TUTase 2 (RET2), catalyze 3' uridylylation of guide RNAs and U-insertions into the mRNAs, respectively. RET1 is also involved in mitochondrial mRNA turnover and participates in numerous heterogeneous complexes; RET2 is an integral part of the 20S editosome, in which it forms a U-insertion subcomplex with zinc finger protein MP81 and RNA editing ligase REL2. Here we report the identification of a third mitochondrial TUTase from Trypanosoma brucei. The mitochondrial editosome-like complex associated TUTase (MEAT1) interacts with a 20S editosome-like particle, effectively substituting the U-insertion subcomplex. MEAT1 and RET2 are mutually exclusive in their respective complexes, which otherwise share several components. Similarly to RET2, MEAT1 is exclusively U-specific in vitro and is active on gapped double-stranded RNA resembling editing substrates. However, MEAT1 does not require a 5' phosphate group on the 3' mRNA cleavage fragment produced by editing endonucleases. The functional RNAi complementation experiments showed that MEAT1 is essential for viability of bloodstream and insect parasite forms. The growth inhibition phenotype in the latter can be rescued by coexpressing an RNAi-resistant gene with double-stranded RNA targeting the endogenous transcript. However, preliminary RNA analysis revealed no gross effects on RNA editing in MEAT1-depleted cells and indicated its possible role in regulating the mitochondrial RNA stability.
| S-EPMC2704088 | biostudies-literature
Project description:The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. We show here that various point mutations to KREPB4 and KREPB5 identify essential domains, and suggest that these proteins do not themselves perform RNase III catalysis. The zinc finger of KREPB4 but not KREPB5 is essential for editosome integrity and parasite viability, and mutation of the RNase III signature motif in KREPB5 prevents integration into editosomes, which is lethal. Isolated TAP-tagged KREPB4 and KREPB5 complexes preferentially associate with components of the deletion subcomplex, providing additional insights into editosome architecture. A new alignment of editosome RNase III sequences from several kinetoplastid species implies that KREPB4 and KREPB5 lack catalytic activity and reveals that the PUF motif is present in the editing endonucleases KREN1, KREN2, and KREN3. The data presented here are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.
| S-EPMC3446712 | biostudies-literature