TbMP42, a Protein Component of the RNA Editing Complex in African Trypanosomes, Has Endo-Exoribonuclease Activity

Brecht, Michael; Niemann, Moritz; Schlüter, Elke; Müller, Ulrich F.; Stuart, Ken; Göringer, H. Ulrich

doi:10.1016/j.molcel.2005.01.018

Cited by 56 publications

(101 citation statements)

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“…Interestingly, expression of LtKREPA2 with disrupted zinc fingers in Leishmania resulted in substantial breakdown of the 20S editosome (54), suggesting that these motifs do have a functional role, possibly in interaction with KREX2 or perhaps in proper folding of KREPA2 itself. The latter would be consistent with the finding that correct folding of recombinant KREPA3, which also contains two zinc finger motifs, required zinc ions (47). Expression of KREPA3 with mutated zinc fingers in T. brucei still resulted in incorporation into editosomes but failed to rescue the lethal effect of depleting endogenous KREPA3 (29).…”

Section: Discussionsupporting

confidence: 86%

“…Eukaryotic SSBs typically form hetero-oligomers with multiple OB-fold domains in which some OB-folds are responsible for nucleic acid binding while others mediate protein-protein interactions, often with multiple partners (45,46). Recombinant KREPA6, KREPA3, and KREPA4 all have been shown to be able to bind RNA in vitro, with a preference for gRNA and oligo(U) sequences (32,34,47). The RNA-binding activity of KREPA3 was mapped to the OB-fold domain (47).…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant KREPA6, KREPA3, and KREPA4 all have been shown to be able to bind RNA in vitro, with a preference for gRNA and oligo(U) sequences (32,34,47). The RNA-binding activity of KREPA3 was mapped to the OB-fold domain (47). In addition, there is evidence that the OB-fold domains of KREPA1 and KREPA2 provide substrate-binding platforms for the enzymatic activities in the deletion and insertion subcomplexes, respectively (25,48).…”

Section: Discussionmentioning

confidence: 99%

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A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

Schnaufer

Park

et al. 2010

Journal of Biological Chemistry

126

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Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ϳ20S on glycerol gradients. These ϳ20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ϳ20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease. Here we present a protein-protein interaction map that was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. One of these proteins, KREPA6, interacts with the OB-fold zinc finger protein in each subcomplex that interconnects their two catalytic proteins. Another OB-fold protein, KREPA3, appears to link to the putative endonuclease subcomplex. These results reveal a physical organization that underlies the coordination of the various catalytic and substrate binding activities within the ϳ20S editosomes during the editing process.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

Schnaufer

Park

et al. 2010

Journal of Biological Chemistry

126

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“…Functional characterization of mitochondrial proteins in trypanosomes revealed three enzymes implicated in U-insertion/deletion RNA editing, TbMP42, TbMP99 and TbMP100. 33,34 TbMP42 displays in vitro exoUase activity leaving 3'-monophosphate ends, which, as in Diplonema, cannot be ligated with the 5' terminus of the downstream pre-mRNA cleavage fragment. In turn, TbMP99 and TbMP100 exhibit 3'-specific nucleotidyl phosphatase activity converting the 3'NMP to a 3' hydroxyl group, which permits pre-mRNA re-ligation.…”

Section: Discussionmentioning

confidence: 99%

RNA-level unscrambling of fragmented genes inDiplonemamitochondria

et al. 2013

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“…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).…”

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confidence: 99%

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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TbMP42, a Protein Component of the RNA Editing Complex in African Trypanosomes, Has Endo-Exoribonuclease Activity

Cited by 56 publications

References 50 publications

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

RNA-level unscrambling of fragmented genes inDiplonemamitochondria

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

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