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

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National Academy of Sciences

RESUMO

The ≈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 ≈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.

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