Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract

Peris, Marian; Simpson, Agda M.; Grunstein, Jeremy; Liliental, Joanna; Frech, Georges C.; Simpson, Larry

doi:10.1016/s0166-6851(96)02795-8

Cited by 39 publications

(47 citation statements)

References 28 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%

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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%

mentioning

confidence: 99%

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 LtREL1 and LtREL2 RNA ligase proteins can be detected by adenylation with [␣ 32 P]ATP (Sabatini and Hajduk 1995;Peris et al 1997). Evidence of an interaction of ATPlabeled LtREL1 and LtREL2 in mitochondrial extract with Ltp26 was provided by the co-IP with anti-Ltp26 antiserum shown in Figure 8B, right panel.…”

Section: Interaction Of the Ltp26/ltp28 Complex With Components Of Edmentioning

confidence: 96%

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

Aphasizhev¹,

Aphasizheva²,

Nelson³

et al. 2003

RNA

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A stable 100-kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologs of gBP27 and gBP29 from Crithidia fasciculata and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins, and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high-molecular-weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60-kD proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L-complex and with the 3 TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.

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“…The core editing complex, known as the L-complex, ''20S complex,'' ''editosome,'' or ''ϳ20S editosome,'' was detected by following the sedimentation and gel filtration of 2 adenylatable RNA ligases using Leishmania tarentolae and Trypanosoma brucei mitochondrial extracts (11,12). The activity sedimented around 20 -25S and migrated as a single band in a native gel (13). A ϳ20S multiprotein complex that showed both U-insertion and U-deletion in vitro activities was also purified from T. brucei mitochondria by column chromatography.…”

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Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria

Li¹,

Hui

et al. 2009

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

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Uridine insertion/deletion RNA editing is a unique form of posttranscriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the core editing complex or ''L (ligase)-complex'' from Leishmania tarentolae mitochondria isolated by the tandem affinity purification procedure (TAP). The purified material, sedimented at 20 -25S, migrated in a blue native gel at 1 MDa and exhibited both precleaved and full-cycle gRNA-mediated U-insertion and U-deletion in vitro activities. The purified L-complex was analyzed by electron tomography to determine the extent of heterogeneity. Three-dimensional structural comparisons of individual particles in the tomograms revealed that a majority of the complexes have a similar shape of a slender triangle. An independent single-particle reconstruction, using a featureless Gaussian ball as the initial model, converged to a similar triangular structure. Another singleparticle reconstruction, using the averaged tomography structure as the initial model, yielded a similar structure. The REL1 ligase was localized on the model to the base of the apex by decoration with REL1-specific IgG. This structure should prove useful for a detailed analysis of the editing reaction.editing ͉ electron microscopy ͉ kinetoplast ͉ Leishmania ͉ trypanosome U ridine (U) insertion/deletion RNA editing is a posttranscriptional process in mitochondria of kinetoplastid protists that involves the modification of mRNA transcripts of mitochondrial cryptogenes by precise insertion and deletion of U residues to create translatable sequences (1-3). We previously proposed a model for the mechanism of this editing process involving a cleavage, addition or deletion of U's and ligation progressing in a 3Ј to 5Ј polarity (4), and this model has been since experimentally validated in almost every detail (5-10).Editing involves multiple multiprotein complexes associated by RNA linkers. The core editing complex, known as the L-complex, ''20S complex,'' ''editosome,'' or ''ϳ20S editosome,'' was detected by following the sedimentation and gel filtration of 2 adenylatable RNA ligases using Leishmania tarentolae and Trypanosoma brucei mitochondrial extracts (11,12). The activity sedimented around 20 -25S and migrated as a single band in a native gel (13). A ϳ20S multiprotein complex that showed both U-insertion and U-deletion in vitro activities was also purified from T. brucei mitochondria by column chromatography. An improved isolation of the L-complex from L. tarentolae was achieved by the tandem affinity purification procedure (TA P) (14); TA P-tagged plasmidexpressed REL1 became incorporated into the L-complex in vivo, allowing isolation of tagged complexes.Fourteen proteins were initially identified by mass spectrometry of the L. tarentolae REL1-TAP pull-down (14). All of the L. tarentolae proteins had homologs in T. brucei, and several additional proteins found in the T. brucei L-complex (15) were later identified in L. tarentolae (Tables S1 and S2).Two 3-prot...

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Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract

Cited by 39 publications

References 28 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

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria

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