Trypanosome RNA Editing Research Articles

RESC14 and RESC8 cooperate to mediate RESC function and dynamics during trypanosome RNA editing.

Mitochondrial transcripts in Trypanosoma brucei require extensive uridine insertion/deletion RNA editing to generate translatable open reading frames. The RNA editing substrate binding complex (RESC) serves as the scaffold that coordinates the protein-protein and protein-RNA interactions during editing. RESC broadly contains two modules termed the guide RNA binding complex (GRBC) and the RNA editing mediator complex (REMC), as well as organizer proteins. How the protein and RNA components of RESC dynamically interact to facilitate editing is not well understood. Here, we examine the roles of organizer proteins, RESC8 and RESC14, in facilitating RESC dynamics. High-throughput sequencing of editing intermediates reveals an overlapping RESC8 and RESC14 function during editing progression across multiple transcripts. Blue native PAGE analysis demonstrates that RESC14 is essential for incorporation of RESC8 into a large RNA-containing complex, while RESC8 is important in recruiting a smaller ribonucleoprotein complex (RNP) to this large complex. Proximity labeling shows that RESC14 is important for stable RESC protein-protein interactions, as well as RESC-RECC associations. Together, our data support a model in which RESC14 is necessary for assembly of editing competent RESC through recruitment of an RNP containing RESC8, GRBC and gRNA to REMC and mRNA.

Targeting RNA Structure to Inhibit Editing in Trypanosomes.

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.

KREH1 RNA helicase activity promotes utilization of initiator gRNAs across multiple mRNAs in trypanosome RNA editing.

Mitochondrial U-indel RNA editing in kinetoplastid protozoa is directed by trans-acting gRNAs and mediated by a holoenzyme with associated factors. Here, we examine the function of the holoenzyme-associated KREH1 RNA helicase in U-indel editing. We show that KREH1 knockout (KO) impairs editing of a small subset of mRNAs. Overexpression of helicase-dead mutants results in expanded impairment of editing across multiple transcripts, suggesting the existence of enzymes that can compensate for KREH1 in KO cells. In depth analysis of editing defects using quantitative RT-PCR and high-throughput sequencing reveals compromised editing initiation and progression in both KREH1-KO and mutant-expressing cells. In addition, these cells exhibit a distinct defect in the earliest stages of editing in which the initiator gRNA is bypassed, and a small number of editing events takes place just outside this region. Wild type KREH1 and a helicase-dead KREH1 mutant interact similarly with RNA and holoenzyme, and overexpression of both similarly disorders holoenzyme homeostasis. Thus, our data support a model in which KREH1 RNA helicase activity facilitates remodeling of initiator gRNA-mRNA duplexes to permit accurate utilization of initiating gRNAs on multiple transcripts.

Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10.

Uridine insertion/deletion editing of mitochondrial mRNAs is a characteristic feature of kinetoplastids, including Trypanosoma brucei. Editing is directed by trans-acting gRNAs and catalyzed by related RNA Editing Core Complexes (RECCs). The non-catalytic RNA Editing Substrate Binding Complex (RESC) coordinates interactions between RECC, gRNA and mRNA. RESC is a dynamic complex comprising GRBC (Guide RNA Binding Complex) and heterogeneous REMCs (RNA Editing Mediator Complexes). Here, we show that RESC10 is an essential, low abundance, RNA binding protein that exhibits RNase-sensitive and RNase-insensitive interactions with RESC proteins, albeit its minimal in vivo interaction with RESC13. RESC10 RNAi causes extensive RESC disorganization, including disruption of intra-GRBC protein–protein interactions, as well as mRNA depletion from GRBC and accumulation on REMCs. Analysis of mitochondrial RNAs at single nucleotide resolution reveals transcript-specific effects: RESC10 dramatically impacts editing progression in pan-edited RPS12 mRNA, but is critical for editing initiation in mRNAs with internally initiating gRNAs, pointing to distinct initiation mechanisms for these RNA classes. Correlations between sites at which editing pauses in RESC10 depleted cells and those in knockdowns of previously studied RESC proteins suggest that RESC10 acts upstream of these factors and that RESC is particularly important in promoting transitions between uridine insertion and deletion RECCs.

Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

SEC–SAXS studies of double-stranded RNAs from trypanosome RNA editing

SEC–SAXS studies of double-stranded RNAs from trypanosome RNA editing

MRB7260 is essential for productive protein-RNA interactions within the RNA editing substrate binding complex during trypanosome RNA editing.

The trypanosome RNA editing substrate binding complex (RESC) acts as the platform for mitochondrial uridine insertion/deletion RNA editing and facilitates the protein–protein and protein–RNA interactions required for the editing process. RESC is broadly comprised of two subcomplexes: GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex). Here, we characterize the function and position in RESC organization of a previously unstudied RESC protein, MRB7260. We show that MRB7260 forms numerous RESC-related complexes, including a novel, small complex with the guide RNA binding protein, GAP1, which is a canonical GRBC component, and REMC components MRB8170 and TbRGG2. RNA immunoprecipitations in MRB7260-depleted cells show that MRB7260 is critical for normal RNA trafficking between REMC and GRBC. Analysis of protein–protein interactions also reveals an important role for MRB7260 in promoting stable association of the two subcomplexes. High-throughput sequencing analysis of RPS12 mRNAs from MRB7260 replete and depleted cells demonstrates that MRB7260 is critical for gRNA exchange and early gRNA utilization, with the exception of the initiating gRNA. Together, these data demonstrate that MRB7260 is essential for productive protein–RNA interactions with RESC during RNA editing.

Trypanosome RNA Editing Mediator Complex proteins have distinct functions in gRNA utilization.

Uridine insertion/deletion RNA editing is an essential process in kinetoplastid parasites whereby mitochondrial mRNAs are modified through the specific insertion and deletion of uridines to generate functional open reading frames, many of which encode components of the mitochondrial respiratory chain. The roles of numerous non-enzymatic editing factors have remained opaque given the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus roles of individual proteins. Here, we examined whole populations of partially edited sequences using high throughput sequencing and a novel bioinformatic platform, the Trypanosome RNA Editing Alignment Tool (TREAT), to elucidate the roles of three proteins in the RNA Editing Mediator Complex (REMC). We determined that the factors examined function in the progression of editing through a gRNA; however, they have distinct roles and REMC is likely heterogeneous in composition. We provide the first evidence that editing can proceed through numerous paths within a single gRNA and that non-linear modifications are essential, generating commonly observed junction regions. Our data support a model in which RNA editing is executed via multiple paths that necessitate successive re-modification of junction regions facilitated, in part, by the REMC variant containing TbRGG2 and MRB8180.

Multiple G-quartet structures in pre-edited mRNAs suggest evolutionary driving force for RNA editing in trypanosomes.

Mitochondrial transcript maturation in African trypanosomes requires a U-nucleotide specific RNA editing reaction. In its most extreme form hundreds of U’s are inserted into and deleted from primary transcripts to generate functional mRNAs. Unfortunately, both origin and biological role of the process have remained enigmatic. Here we report a so far unrecognized structural feature of pre-edited mRNAs. We demonstrate that the cryptic pre-mRNAs contain numerous clustered G-nt, which fold into G-quadruplex (GQ) structures. We identified 27 GQ’s in the different pre-mRNAs and demonstrate a positive correlation between the steady state abundance of guide (g)RNAs and the sequence position of GQ-elements. We postulate that the driving force for selecting G-rich sequences lies in the formation of DNA/RNA hybrid G-quadruplex (HQ) structures between the pre-edited transcripts and the non-template strands of mitochondrial DNA. HQ’s are transcription termination/replication initiation sites and thus guarantee an unperturbed replication of the mt-genome. This is of special importance in the insect-stage of the parasite. In the transcription-on state, the identified GQ’s require editing as a GQ-resolving activity indicating a link between replication, transcription and RNA editing. We propose that the different processes have coevolved and suggest the parasite life-cycle and the single mitochondrion as evolutionary driving forces.

Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry.

Using direct methods starting from random phases, the crystal structure of a 32-base-pair RNA (675 non-H RNA atoms in the asymmetric unit) was determined using only the native diffraction data (resolution limit 1.05 Å) and the computer program SIR2014. The almost three helical turns of the RNA in the asymmetric unit introduced partial or imperfect translational pseudosymmetry (TPS) that modulated the intensities when averaged by the l Miller indices but still escaped automated detection. Almost six times as many random phase sets had to be tested on average to reach a correct structure compared with a similar-sized RNA hairpin (27 nucleotides, 580 non-H RNA atoms) without TPS. More sensitive methods are needed for the automated detection of partial TPS.

High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing.

Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steady-state mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5′, in wild-type Trypanosoma brucei. We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5′ UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences.

Trypanosome RNA editing: the complexity of getting U in and taking U out.

RNA editing, which adds sequence information to RNAs post-transcriptionally, is a widespread phenomenon throughout eukaryotes. The most complex form of this process is the uridine (U) insertion/deletion editing that occurs in the mitochondria of kinetoplastid protists. RNA editing in these flagellates is specified by trans-acting guide RNAs and entails the insertion of hundreds and deletion of dozens of U residues from mitochondrial RNAs to produce mature, translatable mRNAs. An emerging model indicates that the machinery required for trypanosome RNA editing is much more complicated than previously appreciated. A family of RNA editing core complexes (RECCs), which contain the required enzymes and several structural proteins, catalyze cycles of U insertion and deletion. A second, dynamic multiprotein complex, the Mitochondrial RNA Binding 1 (MRB1) complex, has recently come to light as another essential component of the trypanosome RNA editing machinery. MRB1 likely serves as the platform for kinetoplastid RNA editing, and plays critical roles in RNA utilization and editing processivity. MRB1 also appears to act as a hub for coordination of RNA editing with additional mitochondrial RNA processing events. This review highlights the current knowledge regarding the complex molecular machinery involved in trypanosome RNA editing. WIREs RNA 2016, 7:33-51. doi: 10.1002/wrna.1313 For further resources related to this article, please visit the WIREs website.

Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing.

Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2.

The most unlikely places.

The most unlikely places.

Fusion RNAs in crystallographic studies of double-stranded RNA from trypanosome RNA editing.

Head-to-head fusions of two identical double-stranded fragments of RNA can be designed to self-assemble from a single RNA species and form a double-stranded helix with a twofold rotation axis relating the two strands. These symmetrical RNA molecules are more likely to crystallize without end-on-end statistical packing disorder because the two halves of the molecule are identical. This approach can be used to study many fragments of double-stranded RNA or many isolated helical domains from large single-stranded RNAs that may not yet be amenable to high-resolution studies by crystallography or NMR. We used fusion RNAs to study one (the U-helix) of three functional domains formed when guide RNA binds to its cognate pre-edited mRNA from the trypanosome RNA editing system. The U-helix forms when the 3' oligo(U) tail of the guide RNA (gRNA) binds to the purine-rich, pre-edited mRNA upstream from the current RNA editing site. Fusion RNAs 16-and 32-base pairs in length formed crystals that gave diffraction to 1.37 and 1.05 Å respectively. We provide the composition of a fusion RNA crystallization screen and describe the X-ray data collection, structure determination, and refinement of the crystal structures of fusion RNAs.

Structural studies of a double-stranded RNA from trypanosome RNA editing by small-angle X-ray scattering.

We used small-angle X-ray scattering (SAXS) to evaluate the solution structure of a double-stranded RNA with 32 base pairs. We wanted to compare the solution structure to the crystal structure to assess the impact of the crystal lattice on the overall conformation of the RNA. The RNA was designed to self-anneal and form a head-to-head fusion of two identical mRNA/oligo(U) tail domains (the U-helix) from a trypanosome RNA editing substrate formed by the annealing of a guide RNA to a pre-edited mRNA. This substrate is from the U insertion/deletion RNA editing system of trypanosomes. Each strand in the fusion RNA had 16 purines from the pre-mRNA followed by 16 uracils (Us) from the U-tail at the 3' end of the guide RNA. The strands were designed to form a double helix with blunt ends, but each strand had the potential to form hairpins and single-stranded RNA helices. Hairpins could form by the 3' oligouridylate tract folding back to hybridize with the 5' oligopurine tract and forming an intervening loop. Single-stranded helices could form by the stacking of bases in the polypurine tract. Some of the 16 Us 3' to the polypurine tract may have been unstacked and in random coils. Our SAXS studies showed that the RNA formed a mix of single-stranded structures in the absence of MgCl2. In the presence of MgCl2 at concentrations similar to those in the crystal, the solution structure was consistent with the double-stranded, blunt-ended structure, in agreement with the crystal structure. Here we describe the preparation of RNA samples, data collection with an in-house SAXS instrument designed for biological samples, and the processing and modeling of the scattering data.

RNA catalyst as a reporter for screening drugs against RNA editing in trypanosomes.

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based "mix and measure" hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

RNA editing in trypanosomal parasites (232.1)

RNA editing in Trypanosomes is a posttranscriptional process that recodes mitochondrial mRNAs by inserting and deleting uridylates, as specified by gRNAs. The polygenic pre‐mRNA and pre‐gRNA transcripts are processed by several multiprotein complexes; 20S editosomes that serially catalyze RNA cleavage, U addition or removal and ligation and other complexes that add U tails to gRNAs, associate cognate gRNAs and mRNAs and add mRNA poly (A/U) tails. The complexes and accessory factors discriminate among thousands of similar editing sites (ESs) and edit precisely producing the translated mRNAs. The discrimination employs three editosome variants that each have one of three related endonuclease/partner‐protein pairs and different editing site cleavage specificities. The edited mRNAs encode oxidative phosphorylation system components. However, the mRNAs are differentially edited between lifecycle stages by a mechanism that is not yet known. Consequently insect stage T. brucei has a complete oxidative phosphorylation system while the animal stage does not and generates energy by glycolysis. Regulation of RNA editing is integrated within the network of cellular processes that recalibrate metabolic processes and cell surface composition. The animal forms serially express variant surface glycoproteins (VSGs) resulting in antigenic variation while Procyclin surface proteins, not VSGs, are expressed in the insect stage. A future challenge is to elucidate how these multiple cellular processes are integrated. The discovery of editing led to the realization that genomic information can be recoded and the findings that multiple mechanisms of editing exist and are widespread. The exploration of its integration within cellular processes may provide other novel insights.

Mitochondrial RNA editing in trypanosomes: small RNAs in control.

Mitochondrial mRNA editing in trypanosomes is a posttranscriptional processing pathway thereby uridine residues (Us) are inserted into, or deleted from, messenger RNA precursors. By correcting frameshifts, introducing start and stop codons, and often adding most of the coding sequence, editing restores open reading frames for mitochondrially-encoded mRNAs. There can be hundreds of editing events in a single pre-mRNA, typically spaced by few nucleotides, with U-insertions outnumbering U-deletions by approximately 10-fold. The mitochondrial genome is composed of ∼50 maxicircles and thousands of minicircles. Catenated maxi- and minicircles are packed into a dense structure called the kinetoplast; maxicircles yield rRNA and mRNA precursors while guide RNAs (gRNAs) are produced predominantly from minicircles, although varying numbers of maxicircle-encoded gRNAs have been identified in kinetoplastids species. Guide RNAs specify positions and the numbers of inserted or deleted Us by hybridizing to pre-mRNA and forming series of mismatches. These 50-60 nucleotide (nt) molecules are 3' uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Editing reactions of mRNA cleavage, U-insertion or deletion, and ligation are catalyzed by the RNA editing core complex (RECC). To function in mitochondrial translation, pre-mRNAs must further undergo post-editing 3' modification by polyadenylation/uridylation. Recent studies revealed a highly compound nature of mRNA editing and polyadenylation complexes and their interactions with the translational machinery. Here we focus on mechanisms of RNA editing and its functional coupling with pre- and post-editing 3' mRNA modification and gRNA maturation pathways.