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.

• Keywords
• MRP1/2, RNA editing, RNA editing initiation, RNA-binding protein, inhibitor, trypanosome,
• MeSH
• RNA Editing drug effects   genetics   MeSH
• RNA, Guide, Kinetoplastida drug effects   MeSH
• Ligases antagonists & inhibitors   MeSH
• RNA, Messenger genetics   MeSH
• Mitochondrial Proteins genetics   MeSH
• Mitochondria drug effects   genetics   MeSH
• RNA-Binding Proteins genetics   MeSH
• Protozoan Proteins genetics   MeSH
• Recombinant Proteins genetics   MeSH
• RNA, Mitochondrial genetics   MeSH
• RNA, Protozoan genetics   MeSH
• Trypanosoma brucei brucei drug effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gBP21 protein, Trypanosoma brucei MeSH Browser
• gBP25 protein, Trypanosoma brucei MeSH Browser
• RNA, Guide, Kinetoplastida MeSH
• Ligases MeSH
• RNA, Messenger MeSH
• Mitochondrial Proteins MeSH
• RNA-Binding Proteins MeSH
• Protozoan Proteins MeSH
• Recombinant Proteins MeSH
• RNA, Mitochondrial MeSH
• RNA, Protozoan 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.

• Keywords
• RNA editing, mitochondrion, trypanosome,
• MeSH
• Cell Line MeSH
• RNA Editing * MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mitochondrial Proteins physiology   MeSH
• Mitochondria MeSH
• Protozoan Proteins physiology   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• RNA, Messenger MeSH
• Mitochondrial Proteins MeSH
• Protozoan Proteins MeSH

The mitochondrial RNA-binding proteins (MRP) 1 and 2 play a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Here, we report the purification of a high molecular weight protein complex consisting solely of the MRP1 and MRP2 proteins from the mitochondrion of T. brucei. The MRP1/MRP2 complex natively purified from T. brucei and the one reconstituted in Escherichia coli in vivo bind guide (g) RNAs and pre-mRNAs with dissociation constants in the nanomolar range, and efficiently promote annealing of pre-mRNAs with their cognate gRNAs. In addition, the MRP1/MRP2 complex stimulates annealing between two non-cognate RNA molecules suggesting that along with the cognate duplexes, spuriously mismatched RNA hybrids may be formed at some rate in vivo. A mechanism of catalysed annealing of gRNA/pre-mRNA by the MRP1/MRP2 complex is proposed.

• MeSH
• Chromatography MeSH
• RNA Editing MeSH
• Microscopy, Electron MeSH
• RNA, Guide, Kinetoplastida metabolism   ultrastructure   MeSH
• Mitochondrial Proteins * metabolism   physiology   ultrastructure   MeSH
• RNA Precursors metabolism   ultrastructure   MeSH
• Multidrug Resistance-Associated Proteins * chemistry   physiology   MeSH
• RNA-Binding Proteins * metabolism   ultrastructure   MeSH
• Recombinant Proteins pharmacology   MeSH
• RNA, Protozoan genetics   MeSH
• Trypanosoma brucei brucei * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• RNA, Guide, Kinetoplastida MeSH
• Mitochondrial Proteins * MeSH
• RNA Precursors MeSH
• Multidrug Resistance-Associated Proteins * MeSH
• RNA-Binding Proteins * MeSH
• Recombinant Proteins MeSH
• RNA, Protozoan MeSH