APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific Proteins in Trypanosoma brucei

ABSTRACT Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 “proximity proteome” having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified. IMPORTANCE Sleeping sickness is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need for approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei and has allowed identification of proximity proteomes for different flagellar subdomains that cannot be purified. This capacity opens the possibility to study the composition and function of other compartments. We expect this approach may be extended to other eukaryotic pathogens and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilium function is impaired.

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APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei

10.1101/2020.03.09.984815 ◽

2020 ◽

Author(s):

Daniel E. Vélez-Ramírez ◽

Michelle M. Shimogawa ◽

Sunayan Ray ◽

Andrew Lopez ◽

Shima Rayatpisheh ◽

...

Keyword(s):

Membrane Protein ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protein Composition ◽

Principal Component ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Critical Gap ◽

Flagellar Membrane ◽

And Function

ABSTRACTTrypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 “proximity proteome” showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCESleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and has allowed identification of proximity proteomes for different subdomains. This capacity opens the possibility to study the composition and function of other compartments. We further expect that this approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.

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The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00879-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6151-6160 ◽

Cited By ~ 16

Author(s):

Luis Carvalho ◽

Marta Martínez-García ◽

Ignacio Pérez-Victoria ◽

José Ignacio Manzano ◽

Vanessa Yardley ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Antimalarial Drug ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Phase Iii ◽

Content Type ◽

Clinical Phase ◽

Intracellular Ca ◽

Cytoplasmic Content

ABSTRACTThe protozoan parasiteTrypanosoma bruceicauses human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills differentT. bruceispp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca2+, and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significantin vitroactivity againstT. brucei.

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Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽

10.12688/f1000research.10342.2 ◽

2017 ◽

Vol 6 ◽

pp. 683 ◽

Cited By ~ 31

Author(s):

Terry K. Smith ◽

Frédéric Bringaud ◽

Derek P. Nolan ◽

Luisa M. Figueiredo

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protozoan Parasite ◽

Tsetse Fly ◽

Metabolic Reprogramming ◽

Mammalian Host ◽

Insect Vector ◽

Environmental Signals ◽

Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

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Human African trypanosomiasis

Oxford Textbook of Medicine ◽

10.1093/med/9780199204854.003.070810_update_001 ◽

2010 ◽

pp. 1119-1127

Author(s):

August Stich

Keyword(s):

West Africa ◽

East Africa ◽

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Tsetse Flies ◽

African Trypanosomiasis ◽

Tropical Africa

Human African trypanosomiasis (HAT, sleeping sickness) is caused by two subspecies of the protozoan parasite Trypanosoma brucei: T. b. rhodesiense is prevalent in East Africa among many wild and domestic mammals; T. b. gambiense causes an anthroponosis in Central and West Africa. The disease is restricted to tropical Africa where it is transmitted by the bite of infected tsetse flies (...

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Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei

mSphere ◽

10.1128/msphere.00394-17 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 4

Author(s):

Anyango D. Kamina ◽

Daniel Jaremko ◽

Linda Christen ◽

Noreen Williams

Keyword(s):

Trypanosoma Brucei ◽

Ribosome Biogenesis ◽

Sleeping Sickness ◽

5S Rrna ◽

Ribonucleoprotein Particle ◽

Content Type ◽

Assembly Factor ◽

Specific Proteins ◽

First Time ◽

Ribosome Formation

ABSTRACT Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness. Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness.

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Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02065-18 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 2

Author(s):

Antonia Efstathiou ◽

Nicolas Gaboriaud-Kolar ◽

Vassilios Myrianthopoulos ◽

Konstantina Vougogiannopoulou ◽

Ines Subota ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Glycogen Synthase ◽

Intracellular Localization ◽

Protozoan Parasite ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

Content Type ◽

Antitrypanosomal Activity ◽

Half Maximal Effective Concentration

ABSTRACT The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 μM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3′-oxime bearing an extra bulky substituent on the 3′ oxime [(6-BIO-3′-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3′-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo. To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3′-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

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A New NonpolarN-Hydroxy Imidazoline Lead Compound with Improved Activity in a Murine Model of Late-Stage Trypanosoma brucei brucei Infection Is Not Cross-Resistant with Diamidines

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03958-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 890-904 ◽

Cited By ~ 11

Author(s):

Carlos H. Ríos Martínez ◽

Florence Miller ◽

Kayathiri Ganeshamoorthy ◽

Fabienne Glacial ◽

Marcel Kaiser ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Late Stage ◽

Parent Compound ◽

Central Nervous System Infection ◽

Sleeping Sickness ◽

Intrinsic Activity ◽

Cross Resistance ◽

Content Type

ABSTRACTTreatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four newN-hydroxy and 12 newN-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluatedin vitroagainstTrypanosoma brucei rhodesienseandin vivoin murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters,in vitroBBB permeability, and microsomal stability also were determined. TheN-hydroxy imidazoline analogues were the most effectivein vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was foundin vitro, suggesting thatN-hydroxy imidazolines are metabolically stable and have intrinsic activity againstT. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d inT. bruceiwas independent of known drug transporters (i.e.,T. bruceiAT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, theN-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.

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CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00068-13 ◽

2013 ◽

Vol 12 (9) ◽

pp. 1202-1213 ◽

Cited By ~ 11

Author(s):

HoangKim T. Nguyen ◽

Jaspreet Sandhu ◽

Gerasimos Langousis ◽

Kent L. Hill

Keyword(s):

Trypanosoma Brucei ◽

Common Ancestor ◽

Normal Development ◽

Potential Interaction ◽

Content Type ◽

Motility Regulation ◽

Regulatory Complex ◽

Core Components ◽

Insight Into ◽

Flagellar Axoneme

ABSTRACT The eukaryotic flagellum (or cilium) is a broadly conserved organelle that provides motility for many pathogenic protozoa and is critical for normal development and physiology in humans. Therefore, defining core components of motile axonemes enhances understanding of eukaryotic biology and provides insight into mechanisms of inherited and infectious diseases in humans. In this study, we show that component of motile flagella 22 (CMF22) is tightly associated with the flagellar axoneme and is likely to have been present in the last eukaryotic common ancestor. The CMF22 amino acid sequence contains predicted IQ and A TPase a ssociated with a variety of cellular a ctivities (AAA) motifs that are conserved among CMF22 orthologues in diverse organisms, hinting at the importance of these domains in CMF22 function. Knockdown by RNA interference (RNAi) and rescue with an RNAi-immune mRNA demonstrated that CMF22 is required for propulsive cell motility in Trypanosoma brucei . Loss of propulsive motility in CMF22-knockdown cells was due to altered flagellar beating patterns, rather than flagellar paralysis, indicating that CMF22 is essential for motility regulation and likely functions as a fundamental regulatory component of motile axonemes. CMF22 association with the axoneme is weakened in mutants that disrupt the nexin-dynein regulatory complex, suggesting potential interaction with this complex. Our results provide insight into the core machinery required for motility of eukaryotic flagella.

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Diversification of Function by Different Isoforms of Conventionally Shared RNA Polymerase Subunits

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0841 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1293-1301 ◽

Cited By ~ 27

Author(s):

Sara Devaux ◽

Steven Kelly ◽

Laurence Lecordier ◽

Bill Wickstead ◽

David Perez-Morga ◽

...

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protozoan Parasite ◽

Higher Plant ◽

The Core ◽

Form Part ◽

Rna Polymerase Subunits

Eukaryotic nuclei contain three classes of multisubunit DNA-directed RNA polymerase. At the core of each complex is a set of 12 highly conserved subunits of which five—RPB5, RPB6, RPB8, RPB10, and RPB12—are thought to be common to all three polymerase classes. Here, we show that four distantly related eukaryotic lineages (the higher plant and three protistan) have independently expanded their repertoire of RPB5 and RPB6 subunits. Using the protozoan parasite Trypanosoma brucei as a model organism, we demonstrate that these distinct RPB5 and RPB6 subunits localize to discrete subnuclear compartments and form part of different polymerase complexes. We further show that RNA interference-mediated depletion of these discrete subunits abolishes class-specific transcription and hence demonstrates complex specialization and diversification of function by conventionally shared subunit groups.

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RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei

mSphere ◽

10.1128/mspheredirect.00585-17 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 3

Author(s):

Jason Carnes ◽

Suzanne M. McDermott ◽

Kenneth Stuart

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Protozoan Parasite ◽

Tsetse Fly ◽

Accessory Proteins ◽

Rnase Iii ◽

Parasite Life Cycle ◽

Null Cell ◽

Content Type

ABSTRACT Editosomes are the multiprotein complexes that catalyze the insertion and deletion of uridines to create translatable mRNAs in the mitochondria of kinetoplastids. Recognition and cleavage of a broad diversity of RNA substrates in vivo require three functionally distinct RNase III-type endonucleases, as well as five additional editosome proteins that contain noncatalytic RNase III domains. RNase III domains have recently been identified in the editosome accessory proteins KREPB9 and KREPB10, suggesting a role related to editing endonuclease function. In this report, we definitively show that KREPB9 and KREPB10 are not essential in either bloodstream-form parasites (BF) or procyclic-form parasites (PF) by creating null or conditional null cell lines. While preedited and edited transcripts are largely unaffected by the loss of KREPB9 in both PF and BF, loss of KREPB10 produces distinct responses in BF and PF. BF cells lacking KREPB10 also lack edited CYb, while PF cells have increased edited A6, RPS12, ND3, and COII after loss of KREPB10. We also demonstrate that mutation of the RNase III domain of either KREPB9 or KREPB10 results in decreased association with ~20S editosomes. Editosome interactions with KREPB9 and KREPB10 are therefore mediated by the noncatalytic RNase III domain, consistent with a role in endonuclease specialization in Trypanosoma brucei. IMPORTANCE Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in T. brucei generates mature mitochondrial mRNAs. Editing is essential for survival in mammalian hosts and tsetse fly vectors and is differentially regulated during the parasite life cycle. Three multiprotein “editosomes,” typified by exclusive RNase III endonucleases that act at distinct sites, catalyze editing. Here, we show that editosome accessory proteins KREPB9 and KREPB10 are not essential for mammalian blood- or insect-form parasite survival but have specific and differential effects on edited RNA abundance in different stages. We also characterize KREPB9 and KREPB10 noncatalytic RNase III domains and show they are essential for editosome association, potentially via dimerization with RNase III domains in other editosome proteins. This work enhances the understanding of distinct editosome and accessory protein functions, and thus differential editing, during the parasite life cycle and highlights the importance of RNase III domain interactions to editosome architecture.

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