RESUMO
The approximately 20S 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 approximately 16 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.
Assuntos
Carbono-Oxigênio Ligases/metabolismo , Leishmania/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Edição de RNA/fisiologia , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Baculoviridae , Eletroforese , Escherichia coli , Teste de Complementação Genética , Vetores Genéticos , Ligases , Mitocôndrias/fisiologia , Oligonucleotídeos , Interferência de RNA , Trypanosoma brucei brucei/fisiologiaRESUMO
A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.
Assuntos
Leishmania/enzimologia , Mitocôndrias/enzimologia , Edição de RNA , RNA Nucleotidiltransferases/isolamento & purificação , RNA Nucleotidiltransferases/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Cromatografia por Troca Iônica , Leishmania/citologia , Leishmania/genética , Magnésio/metabolismo , Mitocôndrias/química , Dados de Sequência Molecular , Peso Molecular , RNA/genética , RNA/metabolismo , RNA Nucleotidiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/genéticaRESUMO
A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing- or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3' TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids.