The cryo-EM structure of trypanosome 3-methylcrotonyl-CoA carboxylase provides mechanistic and dynamic insights into its enzymatic function.

3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic role of MCC, deficiencies in this enzyme lead to organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each α- and ß-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in a non-filamentous state at 2.4 Å resolution. Biotin is covalently bound to the biotin carboxyl carrier protein domain of α-subunits and positioned in a non-canonical pocket near the active site of neighboring ß-subunit dimers. Moreover, flexibility of key residues at α-subunit interfaces and loops enables pivoting of α-subunit trimers to partly reduce the distance between α- and ß-subunit active sites, required for MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and to assist drug discovery against trypanosome infections.

Structural basis of gRNA stabilization and mRNA recognition in trypanosomal RNA editing.

In Trypanosoma brucei, the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)-programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo-electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA-binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome's catalytic modality.

A new class of antiparasitic drugs.

Cyanotriazole compounds "poison" topoisomerase II of pathogenic trypanosomatids.

CryoEM reveals oligomeric isomers of a multienzyme complex and assembly mechanics.

Propionyl-CoA carboxylase (PCC) is a multienzyme complex consisting of up to six α-subunits and six ß-subunits. Belonging to a metabolic pathway converging on the citric acid cycle, it is present in most forms of life and irregularities in its assembly lead to serious illness in humans, known as propionic acidemia. Here, we report the cryogenic electron microscopy (cryoEM) structures and assembly of different oligomeric isomers of endogenous PCC from the parasitic protozoan Leishmania tarentolae (LtPCC). These structures and their statistical distribution reveal the mechanics of PCC assembly and disassembly at equilibrium. We show that, in solution, endogenous LtPCC ß-subunits form stable homohexamers, to which different numbers of α-subunits attach. Sorting LtPCC particles into seven classes (i.e., oligomeric formulae α0ß6, α1ß6, α2ß6, α3ß6, α4ß6, α5ß6, α6ß6) enables formulation of a model for PCC assembly. Our results suggest how multimerization regulates PCC enzymatic activity and showcase the utility of cryoEM in revealing the statistical mechanics of reaction pathways.

tRNATyr has an unusually short half-life in Trypanosoma brucei.

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.

Discovery, structure, and function of filamentous 3-methylcrotonyl-CoA carboxylase.

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent mitochondrial enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments, and determined the structures of different filament regions at 3.4, 3.9, and 7.3 Å resolution using cryoEM. α6ß6 LtMCCs assemble in a twisted-stacks architecture, manifesting as supramolecular rods up to 400 nm. Filamentous LtMCCs bind biotin non-covalently and lack coenzyme A. Filaments elongate by stacking α6ß6 LtMCCs onto the exterior α-trimer of the terminal LtMCC. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the function of polymerization in the carboxylase superfamily and beyond.

CTS tag-based methods for investigating mitochondrial RNA modification factors in Trypanosoma brucei.

Unicellular parasite Trypanosoma brucei maintains an elaborate mitochondrial mRNA processing pathway including 3'-5' exonucleolytic trimming of primary precursors, 5' and 3' modifications, and, in most cases, massive U-insertion/deletion editing. Whereas the role of editing in restoring protein coding sequence is apparent, recent developments suggest that terminal modifications are equally critical for generating a stable translationally competent messenger. The enzymatic activities responsible for 5' pyrophosphate hydrolysis, 3' adenylation and uridylation, and 3'-5' decay are positively and negatively regulated by pentatricopeptide repeat-containing (PPR) proteins. These sequence-specific RNA binding factors typically contain arrays of 35-amino acid repeats each of which recognizes a single nucleotide. Here, we introduce a combinatorial CTS affinity tag, which underlies a suite of methods for PPR proteins purification, in vivo RNA binding sites mapping and sub-cellular localization studies. These approaches should be applicable to most trypanosomal RNA binding proteins.

Mitochondrial RNA quality control in trypanosomes.

Unicellular parasites Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a spectrum of diseases that jeopardize public health and afflict the economy in sub-Saharan Africa. These hemoflagellates are distinguished by a single mitochondrion, which contains a kinetoplast nucleoid composed of DNA and histone-like proteins. Kinetoplast DNA (kDNA) represents a densely packed network of interlinked relaxed circular molecules: a few ~23-kb maxicircles encoding ribosomal RNAs (rRNAs) and proteins, and approximately 5,000 1-kb minicircles bearing guide RNA (gRNA) genes. The transcription start site defines the mRNA's 5' terminus while the primary RNA is remodeled into a monocistronic messenger by 3'-5' exonucleolytic trimming, 5' and 3' end modifications, and, in most cases, by internal U-insertion/deletion editing. Ribosomal and guide RNA precursors are also trimmed, and the processed molecules are uridylated. For 35 years, mRNA editing has attracted a major effort, but more recently the essential pre- and postediting processing and turnover events have been discovered and the key effectors have been identified. Among these, pentatricopeptide repeat (PPR) RNA binding proteins emerged as conduits coupling modifications of mRNA termini with internal sequence changes introduced by editing. Among 39 annotated PPRs, 20 belong to ribosomal subunits or assembly intermediates, four function as polyadenylation factors, a single factor directs 5' mRNA modification, and one protein is found in F1-ATPase. Nuclear and mitochondrial RNases P consist of a single PPR polypeptide, PRORP1 and PROP2, respectively. Here, we review PPR-mediated mitochondrial processes and discuss their potential roles in mRNA maturation, quality control, translational activation, and decay. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Processing > 3' End Processing RNA Processing > RNA Editing and Modification.

Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei.

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes.

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei.

In Trypanosoma brucei, most mitochondrial mRNAs undergo editing, and 3' adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: pre-editing addition of the short (A) tail protects the edited transcript against 3'-5' degradation, while post-editing A/U-tailing renders mRNA competent for translation. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3' modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes mRNA degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point likely prevents translation of incompletely edited mRNAs. We also find that RNA editing substrate binding complex (RESC) mediates the interaction between the 5' end-bound pyrophosphohydrolase MERS1 and 3' end-associated KPAF4 to enable mRNA circularization. This event appears to be critical for edited mRNA stability.

Transcription initiation defines kinetoplast RNA boundaries.

Mitochondrial genomes are often transcribed into polycistronic RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that in Trypanosoma brucei the monophosphorylated 5' ends of functional molecules typify precursor partitioning by an unknown endonuclease. On the contrary, we demonstrate that individual mRNAs and rRNAs are independently synthesized as 3'-extended precursors. The transcription-defined 5' terminus is converted into a monophosphorylated state by the pyrophosphohydrolase complex, termed the "PPsome." Composed of the MERS1 NUDIX enzyme, the MERS2 pentatricopeptide repeat RNA-binding subunit, and MERS3 polypeptide, the PPsome binds to specific sequences near mRNA 5' termini. Most guide RNAs lack PPsome-recognition sites and remain triphosphorylated. The RNA-editing substrate-binding complex stimulates MERS1 pyrophosphohydrolase activity and enables an interaction between the PPsome and the polyadenylation machinery. We provide evidence that both 5' pyrophosphate removal and 3' adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3'-5' exonucleolytic trimming defines the mRNA 3' end before adenylation. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.

PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes.

In Trypanosoma brucei, most mitochondrial mRNAs undergo internal changes by RNA editing and 3' end modifications. The temporally separated and functionally distinct modifications are manifested by adenylation prior to editing, and by post-editing extension of a short A-tail into a long A/U-heteropolymer. The A-tail stabilizes partially and fully edited mRNAs, while the A/U-tail enables mRNA binding to the ribosome. Here, we identify an essential pentatricopeptide repeat-containing RNA binding protein, kinetoplast polyadenylation factor 3 (KPAF3), and demonstrate its role in protecting pre-mRNA against degradation by the processome. We show that KPAF3 recruits KPAP1 poly(A) polymerase to the 3' terminus, thus leading to pre-mRNA stabilization, or decay depending on the occurrence and extent of editing. In vitro, KPAF3 stimulates KPAP1 activity and inhibits mRNA uridylation by RET1 TUTase. Our findings indicate that KPAF3 selectively directs pre-mRNA toward adenylation rather than uridylation, which is a default post-trimming modification characteristic of ribosomal and guide RNAs. As a quality control mechanism, KPAF3 binding ensures that mRNAs entering the editing pathway are adenylated and, therefore, competent for post-editing A/U-tailing and translational activation.

Constructive edge of uridylation-induced RNA degradation.

RNA uridylation is a significant transcriptome-shaping factor in protists, fungi, metazoans, and plants. The 3' U-additions are catalyzed by terminal uridyltransferases (TUTases), a diverse group of enzymes that along with non-canonical poly(A) polymerases form a distinct group in the superfamily of DNA polymerase ß-like nucleotidyl transferases. Within and across studied organisms and subcellular compartments, TUTases differ in nucleotide triphosphate selectivity, interacting partners, and RNA targets. A general premise linking RNA uridylation to 3'-5' degradation received support from several studies of small RNAs and mRNA turnover. However, recent work on kinetoplastid protists typified by Trypanosoma brucei provides evidence that RNA uridylation may play a more nuanced role in generating functional small RNAs. In this pathogen's mitochondrion, most mRNAs are internally edited by U-insertions and deletions, and subjected to 3' adenylation/uridylation; guide RNAs (gRNAs) required for editing are U-tailed. The prominent role of uridylation in mitochondrial RNA metabolism stimulated identification of the first TUTase, RNA editing TUTase 1 (RET1). Here we discuss functional studies of mitochondrial uridylation in trypanosomes that have revealed an unorthodox pathway of small RNA biogenesis. The current model accentuates physical coupling of RET1 and 3'-5' RNase II/RNB-type exonuclease DSS1 within a stable complex termed the mitochondrial 3' processome (MPsome). In the confines of this complex, RET1 initially uridylates a long precursor to activate its 3'-5' degradation by DSS1, and then uridylates trimmed guide RNA to disengage the processing complex from the mature molecule. We also discuss a potential role of antisense transcription in the MPsome pausing at a fixed distance from gRNA's 5' end. This step likely defines the mature 3' end by enabling kinetic competition between TUTase and exonuclease activities.

RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting.

Terminal uridyltransferases (TUTases) execute 3' RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3' processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3' processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets.

Investigating RNA editing factors from trypanosome mitochondria.

Mitochondrial U-insertion/deletion mRNA editing is carried out by two principal multiprotein assemblies, enzymatic RNA editing core (RECC) and RNA editing substrate binding (RESC) complexes, and a plethora of auxiliary factors. An integral part of mitochondrial gene expression, editing receives inputs from primary mRNA and gRNA precursor processing pathways, and generates substrates for mRNA polyadenylation and translation. Although nearly all RECC-embedded enzymes have been implicated in specific editing reactions, the majority of proteins that populate the RESC are also essential for generating edited mRNAs. However, lack of recognizable motifs in RESC subunits limits the prowess of bioinformatics in guiding biochemical experiments and elucidating their specific biological functions. In this chapter, we describe a generic workflow for investigating mitochondrial mRNA editing in Trypanosoma brucei and focus on several methods that proved instrumental is assigning definitive functions to editing factors lacking known signature sequences.

Antisense Transcripts Delimit Exonucleolytic Activity of the Mitochondrial 3' Processome to Generate Guide RNAs.

Small, noncoding RNA biogenesis typically involves cleavage of structured precursor by RNase III-like endonucleases. However, guide RNAs (gRNAs) that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5' triphosphate characteristic of the transcription initiation and possess a U-tail indicative of 3' processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase, DSS1 3'-5' exonuclease, and three additional subunits. This complex, termed mitochondrial 3' processome (MPsome), is responsible for primary uridylation of ∼800 nt gRNA precursors, their processive degradation to a mature size of 40-60 nt, and secondary U-tail addition. Both strands of the gRNA gene are transcribed into sense and antisense precursors of similar lengths. Head-to-head hybridization of these transcripts blocks symmetrical 3'-5' degradation at a fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5' extremity of a primary molecule by uridylation-induced, antisense transcription-controlled 3'-5' exonucleolytic degradation.

U-Insertion/Deletion mRNA-Editing Holoenzyme: Definition in Sight.

RNA editing is a process that alters DNA-encoded sequences and is distinct from splicing, 5' capping, and 3' additions. In 30 years since editing was discovered in mitochondria of trypanosomes, several functionally and evolutionarily unrelated mechanisms have been described in eukaryotes, archaea, and viruses. Editing events are predominantly post-transcriptional and include nucleoside insertions and deletions, and base substitutions and modifications. Here, we review the mechanism of uridine insertion/deletion mRNA editing in kinetoplastid protists typified by Trypanosoma brucei. This type of editing corrects frameshifts, introduces translation punctuation signals, and often adds hundreds of uridines to create protein-coding sequences. We focus on protein complexes responsible for editing reactions and their interactions with other elements of the mitochondrial gene expression pathway.

Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes.

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3' adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation.

RNA binding and core complexes constitute the U-insertion/deletion editosome.

Enzymes embedded into the RNA editing core complex (RECC) catalyze the U-insertion/deletion editing cascade to generate open reading frames in trypanosomal mitochondrial mRNAs. The sequential reactions of mRNA cleavage, U-addition or removal, and ligation are directed by guide RNAs (gRNAs). We combined proteomic, genetic, and functional studies with sequencing of total and complex-bound RNAs to define a protein particle responsible for the recognition of gRNAs and pre-mRNA substrates, editing intermediates, and products. This approximately 23-polypeptide tripartite assembly, termed the RNA editing substrate binding complex (RESC), also functions as the interface between mRNA editing, polyadenylation, and translation. Furthermore, we found that gRNAs represent only a subset of small mitochondrial RNAs, and yet an inexplicably high fraction of them possess 3' U-tails, which correlates with gRNA's enrichment in the RESC. Although both gRNAs and mRNAs are associated with the RESC, their metabolic fates are distinct: gRNAs are degraded in an editing-dependent process, whereas edited mRNAs undergo 3' adenylation/uridylation prior to translation. Our results demonstrate that the well-characterized editing core complex (RECC) and the RNA binding particle defined in this study (RESC) typify enzymatic and substrate binding macromolecular constituents, respectively, of the ∼40S RNA editing holoenzyme, the editosome.