A Deletion Site Editing Endonuclease in Trypanosoma brucei

Trotter, James R.; Ernst, Nancy Lewis; Carnes, Jason; Panicucci, Brian; Stuart, Kenneth

doi:10.1016/j.molcel.2005.09.016

Cited by 78 publications

(124 citation statements)

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“…Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

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confidence: 99%

The Architecture of Trypanosoma brucei editosomes

McDermott

Luo

Carnes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra-and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.cross-linking | proteomics | Trypanosoma brucei | RNA editing | editosome T he kinetoplastid Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in subSaharan Africa. Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA, which is composed of interlocked DNA maxicircles and minicircles (1, 2). Several identical maxicircles encode two ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs) for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing (3-9). RNA editing was first described in kinetoplastids (6,8,9), where it is essential (10) and involves insertion and deletion of uridines (Us) to generate translatable mitochondrial transcripts. The sequence information for editing is specified by ∼60-nucleotide guide RNAs (gRNAs) that are encoded in thousands of heterogeneous minicircles (11)(12)(13)(14)(15)(16)(17). Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).There are three similar versions of ∼20S editosomes that have a common set of 12 proteins (SI Appendix, Fig. S1 and Table S1). However, they are compositionally and functionally distinct in that each...

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confidence: 99%

The Architecture of Trypanosoma brucei editosomes

McDermott

Luo

Carnes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2). However, recombinant proteins were enzymatically inactive (18,19).In this study, we provide both indirect and direct evidence for a role of the MP90 L-complex protein in the initial cleavage at preedited mRNA U-deletion editing sites, and we show that a full-round cycle of in vitro U-deletion editing can be reconstituted with just three recombinant proteins: REX1, REL1, and MP90. MP90 has been functionally renamed as RNA editing nuclease 1 (REN1).…”

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confidence: 70%

“…In line with suggestions for nomenclature of editing proteins (2,18), the MP90 endoribonuclease is designated REN1. The L-complex 20S gradient-fraction cleavage activity was eliminated by RNAi down-regulation of expression of TbREN1 in procyclic T. brucei.…”

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confidence: 99%

“…Trotter et al (18) and Carnes et al (19) recently provided in vivo evidence for a specific role in cleavage at mRNA U-deletion and U-insertion editing sites for, respectively, the essential TbMP90 and TbMP61 RNase III motif-containing proteins. It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2).…”

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Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

Kang

Gao

Rogers

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Uridine (U)-insertion͞deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model Udeletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNAediting nuclease 1.mitochondria involves the participation of at least three RNAlinked multiprotein complexes, the Ϸ19-polypeptide ligasecontaining core editing complex (L-complex), the mitochondrial RNA-binding protein (MRP) complex, and the RNA editing 3Ј terminal uridylyl transferase (TUTase) (RET)1 complex(es) (1-3). L-complex proteins that have been expressed in active form and characterized include the RNA editing ligases 1 and 2 (REL1 and REL2) (4-7), the RNA editing 3Ј-5Ј U-specific exonucleases (REX1 and REX2) (8-10), and the RET2Ј3Ј TUTase (11-13). Characterization of the protein components of the L-complex led to several nuclease candidates: LC6A, or MP61, MP90, and MP67 contain RNase III motifs, and LC8, or MP44, has a highly diverged RNase III motif (11,(14)(15)(16). Although TbMP90 and TbMP61 were stated in recent reviews (1, 16) to also contain double-strand RNA-binding motifs, such motifs are not readily identifiable (L.S., unpublished data). In addition, the MP42 L-complex protein, which has only zinc finger (ZnF C2H2) and single-strand RNA-binding (SSB) motifs, nevertheless exhibited both exonuclease and endonuclease activities, but the activities identified did not show the required specificity, and the role of this protein remains an open question (17). Trotter et al. (18) and Carnes et al. (19) recently provided in vivo evidence for a specific role in cleavage at mRNA U-deletion and U-insertion editing sites for, respectively, the essential TbMP90 and TbMP61 RNase III motif-containing proteins. It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2). However, recombinant proteins were enzymatically inactive (18,19).In this study, we provide both indirect and direct evidence for a role of the MP90 L-complex protein in the initial cleavage at preedited mRNA U-deletion editing sites, and we sh...

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

2015

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RNA editing, which adds sequence information to RNAs posttranscriptionally, 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 multi-protein 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.

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A Deletion Site Editing Endonuclease in Trypanosoma brucei

Cited by 78 publications

References 50 publications

The Architecture of Trypanosoma brucei editosomes

The Architecture of Trypanosoma brucei editosomes

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

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

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