Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria

Rusché, Laura N.; Cruz‐Reyes, Jorge; Piller, Kenneth J.; Sollner‐Webb, Barbara

doi:10.1093/emboj/16.13.4069

Cited by 159 publications

(357 citation statements)

References 38 publications

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“…The recombinant ligases were incubated with the mitochondrial lysate, which was then fractionated in a glycerol gradient to separate the 20S L-complex (12,13,17). Both homologous T. brucei recombinant ligases and heterologous recombinant ligases from L. tarentolae were equally effective in this reconstitution reaction.…”

Section: Discussionmentioning

confidence: 99%

“…RET1 has been shown to be involved in the addition of nonencoded Us to the 3Ј ends of guide RNAs (11). The RNA ligase-containing L-complex was initially detected as an ␣-32 P-ATP-labeled particle sedimenting Ϸ20S in glycerol gradients and migrating as a single band with an approximate size of 1,200-2,000 kDa in native gels (12,13). This complex has been labeled the editosome but it seems appropriate to reserve this nomenclature for the entire RNA͞protein supercomplex once it is more defined.…”

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confidence: 99%

“…This complex has been labeled the editosome but it seems appropriate to reserve this nomenclature for the entire RNA͞protein supercomplex once it is more defined. The L-complex has been isolated from Trypanosoma brucei and Leishmania tarentolae mitochondria by column chromatography (13)(14)(15)(16) and by tandem affinity purification (TAP) chromatography (17). At least 16 protein components have been identified, including the two RNA-editing ligases (RELs) (REL1 and REL2), a second 3Ј TUTase (RET2), three to four RNase III-motif proteins, three related zinc finger-motif proteins, two AP-endo-exonucleasephosphatase-motif proteins, two RNA binding-motif proteins, and several proteins lacking identifiable motifs.…”

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confidence: 99%

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Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

Gao

Simpson

Kang

et al. 2005

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

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The Ϸ20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains Ϸ16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.editing ͉ RNA-editing ligase 1 ͉ RNA-editing ligase 2 ͉ trypanosome S everal macromolecular complexes have been identified that are involved in U-insertion͞deletion RNA editing in trypanosomatid mitochondria (1-3). The mitochondrial RNA-binding protein complex contains two small RNA-binding proteins and three associated proteins, and may possibly be involved in the initial annealing of the guide RNA and the mRNA (4-9). The RNAediting terminal uridylyl transferase 1 (TUTase 1) (RET1) complex contains the RET1 tetramer and one or more unidentified components (10). RET1 has been shown to be involved in the addition of nonencoded Us to the 3Ј ends of guide RNAs (11). The RNA ligase-containing L-complex was initially detected as an ␣-32 P-ATP-labeled particle sedimenting Ϸ20S in glycerol gradients and migrating as a single band with an approximate size of 1,200-2,000 kDa in native gels (12,13). This complex has been labeled the editosome but it seems appropriate to reserve this nomenclature for the entire RNA͞protein supercomplex once it is more defined. The L-complex has been isolated from Trypanosoma brucei and Leishmania tarentolae mitochondria by column chromatography (13-16) and by tandem affinity purification (TAP) chromatography (17). At least 16 protein components have been identified, including the two RNA-editing ligases (RELs) (REL1 and REL2), a second 3Ј TUTase (RET2), three to four RNase III-motif proteins, three related zinc finger-motif proteins, two AP-endo-exonucleasephosphatase-motif proteins, two RNA binding-motif proteins, and several proteins lacking identifiable motifs.The two ligases are localized in subcomplexes that have been partially defined by coimmunoprecipitation experiments and...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

Gao

Simpson

Kang

et al. 2005

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

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“…The same system also supports editing of a NADH dehydrogenase 7 (ND7)-ATPase 6 chimeric mRNA (3). However, a complete editing cycle of cytochrome b mRNA has not yet been demonstrated with these extracts, even though products are formed in the reaction that are consistent with the expected editing intermediates (5). Leishmania tarentolae mitochondrial extracts support gRNA-directed U-insertions into the first editing site of ND7 mRNA, but the guiding of insertions into a cytochrome b transcript could likewise not be demonstrated with this system (6).…”

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Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

Oppegard¹,

Kabb

Connell

2000

Journal of Biological Chemistry

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The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created only after the addition or deletion of specific uridines. Although in vitro systems have been valuable in characterizing the editing mechanism, only a limited number of mRNAs are accurately edited in vitro. We demonstrate here that in vitro editing of cytochrome b mRNA is inhibited by an A-U sequence present on both the 5-untranslated sequence and on a cytochrome b guide RNA. Mutation of the sequence on the guide RNA stimulates directed editing and results in the loss of binding to at least one component within the editing extract. Mutation of the sequence on the mRNA increases the accuracy of the editing. Evidence is provided that suggests the A-U sequence interacts with the editing machinery both in vitro and in vivo.

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“…wrote the paper. LC1 MP81 II KREPA1 LC2 MP100 I KREPC1 REX1 LC3 MP99 KREPC2 REX2 LC4 MP63 III KREPA2 LC5 MP46 KREPB4 LC6a MP61 KREPB3 REN2 LC6b MP57 KRET2 RET2 LC7a MP52 IV KREL1 REL1 LC7b MP42 VI KREPA3 LC7c MP49 KREPB6 LC8 MP44 KREPB5 LC9 MP48 V KREL2 REL2 LC10 MP24 KREPA4 LC11 MP18 VII KREPA6 MP90 MP90 KREPB1 REN1 MP67 MP67 KREPB2 References: LC (11), MP (40), Band (41), and KREP (1).…”

Section: Usedmentioning

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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Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria

Cited by 159 publications

References 38 publications

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

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

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