Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3' to 5' progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

• MeSH
• editace RNA genetika   MeSH
• guide RNA, Kinetoplastida genetika   MeSH
• messenger RNA genetika   MeSH
• mitochondrie genetika   MeSH
• protozoální proteiny genetika   MeSH
• RNA mitochondriální genetika   MeSH
• RNA protozoální genetika   MeSH
• stabilita RNA genetika   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• guide RNA, Kinetoplastida MeSH
• messenger RNA MeSH
• mitochondrial messenger RNA MeSH Prohlížeč
• protozoální proteiny MeSH
• RNA mitochondriální MeSH
• RNA protozoální MeSH

The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.

• Klíčová slova
• MRP1/2, RNA editing, RNA editing initiation, RNA-binding protein, inhibitor, trypanosome,
• MeSH
• editace RNA účinky léků   genetika   MeSH
• guide RNA, Kinetoplastida účinky léků   MeSH
• ligasy antagonisté a inhibitory   MeSH
• messenger RNA genetika   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie účinky léků   genetika   MeSH
• proteiny vázající RNA genetika   MeSH
• protozoální proteiny genetika   MeSH
• rekombinantní proteiny genetika   MeSH
• RNA mitochondriální genetika   MeSH
• RNA protozoální genetika   MeSH
• Trypanosoma brucei brucei účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gBP21 protein, Trypanosoma brucei MeSH Prohlížeč
• gBP25 protein, Trypanosoma brucei MeSH Prohlížeč
• guide RNA, Kinetoplastida MeSH
• ligasy MeSH
• messenger RNA MeSH
• mitochondriální proteiny MeSH
• proteiny vázající RNA MeSH
• protozoální proteiny MeSH
• rekombinantní proteiny MeSH
• RNA mitochondriální MeSH
• RNA protozoální MeSH

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.

• Klíčová slova
• RNA decay, RNA editing, Trypanosoma, kinetoplast, mitochondria, polyadenylation,
• MeSH
• editace RNA fyziologie   MeSH
• RNA mitochondriální genetika   metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• RNA mitochondriální MeSH
• RNA protozoální MeSH

Protein structure determines biological function. Accurately conceptualizing 3D protein/ligand structures is thus vital to scientific research and education. Virtual reality (VR) enables protein visualization in stereoscopic 3D, but many VR molecular-visualization programs are expensive and challenging to use; work only on specific VR headsets; rely on complicated model-preparation software; and/or require the user to install separate programs or plugins. Here we introduce ProteinVR, a web-based application that works on various VR setups and operating systems. ProteinVR displays molecular structures within 3D environments that give useful biological context and allow users to situate themselves in 3D space. Our web-based implementation is ideal for hypothesis generation and education in research and large-classroom settings. We release ProteinVR under the open-source BSD-3-Clause license. A copy of the program is available free of charge from http://durrantlab.com/protein-vr/, and a working version can be accessed at http://durrantlab.com/pvr/.

• MeSH
• internet * MeSH
• konformace proteinů MeSH
• proteiny * chemie   ultrastruktura   MeSH
• virtuální realita * MeSH
• výpočetní biologie metody   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny * MeSH

MRP1/2 is a heteromeric protein complex that functions in the trypanosomatid mitochondrion as part of the RNA editing machinery, which facilitates multiple targeted insertions and deletions of uridines. MRP1/2 was shown to interact with MRB8170, which initiates RNA editing by marking pre-edited mRNAs, while TbRGG2 is required for its efficient progression on pan-edited mRNAs. Both MRP1/2 and TbRGG2 are capable of modulating RNA-RNA interactions in vitro. As determined by using iCLIP and RIP-qPCR, RNAs bound to MRP1/2 are characterized and compared with those associated with MRB8170 and TbRGG2. We provide evidence that MRP1 and MRB8170 have correlated binding and similar RNA crosslinking peak profiles over minimally and never-edited mRNAs. Our results suggest that MRP1 assists MRB8170 in RNA editing on minimally edited mRNAs.

• Klíčová slova
• RNA binding proteins, RNA editing, iCLIP, mitochondrion, ribonuclear protein, trypanosome,
• MeSH
• editace RNA MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA mitochondriální genetika   metabolismus   MeSH
• Trypanosoma genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gBP21 protein, Trypanosoma brucei MeSH Prohlížeč
• messenger RNA MeSH
• mitochondrial messenger RNA MeSH Prohlížeč
• proteiny vázající RNA MeSH
• protozoální proteiny MeSH
• RNA mitochondriální MeSH

• MeSH
• lidé MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteom analýza   metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• stadia vývoje fyziologie   MeSH
• Trypanosoma brucei brucei růst a vývoj   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom MeSH
• protozoální proteiny MeSH

The mitochondrial (mt) FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF), but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF), which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm). Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1) binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1), but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• hydrolýza MeSH
• inhibitory enzymů metabolismus   MeSH
• protonové ATPasy metabolismus   MeSH
• regulace genové exprese * MeSH
• Trypanosoma brucei brucei enzymologie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• inhibitory enzymů MeSH
• protonové ATPasy MeSH

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.

• Klíčová slova
• RNA editing, mitochondrion, trypanosome,
• MeSH
• buněčné linie MeSH
• editace RNA * MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondriální proteiny fyziologie   MeSH
• mitochondrie MeSH
• protozoální proteiny fyziologie   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• messenger RNA MeSH
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• ferredoxiny metabolismus   MeSH
• frataxin MeSH
• jaderné lokalizační signály MeSH
• lyasy štěpící vazby C-S chemie   genetika   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• proteiny asociované s jadernou matrix chemie   genetika   metabolismus   MeSH
• proteiny vázající železo metabolismus   MeSH
• protozoální proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• Trypanosoma brucei brucei enzymologie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteine desulfurase MeSH Prohlížeč
• ferredoxiny MeSH
• jaderné lokalizační signály MeSH
• lyasy štěpící vazby C-S MeSH
• mitochondriální proteiny MeSH
• proteiny asociované s jadernou matrix MeSH
• proteiny vázající železo MeSH
• protozoální proteiny MeSH

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K(+) accumulation in the matrix.

• Klíčová slova
• Bioenergetics, Letm1, Mitochondria, Potassium Transport, Translation, Trypanosome,
• MeSH
• antibakteriální látky farmakologie   MeSH
• draslík metabolismus   MeSH
• fenotyp MeSH
• homeostáza MeSH
• kationty MeSH
• lidé MeSH
• membránový potenciál mitochondrií MeSH
• mitochondriální proteiny chemie   metabolismus   fyziologie   MeSH
• mitochondrie metabolismus   MeSH
• proteosyntéza MeSH
• protozoální proteiny chemie   metabolismus   fyziologie   MeSH
• průtoková cytometrie metody   MeSH
• RNA interference MeSH
• testy genetické komplementace MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• draslík MeSH
• kationty MeSH
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH