In plant mitochondria, gene expression of translatable mRNAs is a complex process with two critical steps, RNA editing and splicing. We studied the role of RNA editing on non-coding regions of the mat-r-nad1e-nad5c transcript from wheat mitochondria. This RNA contains two trans-introns, 3'-nad1-I4 and 3'-nad5-I2, involved in different trans-splicing events, ensuring the association of nad1d-nad1e and nad5b-nad5c exons from nad1 and nad5 mRNAs respectively. The C-to-U editing changes studied here affect homologous positions on 3'-nad1-I4 and 3'-nad5-I2. It is proposed that these base changes are necessary to place an Adenosine residue in a bulging conformation characteristic of domain VI (D6) from group II introns. In this work, we investigated the role of RNA editing events on 3'-nad1-I4 and 3'-nad5-I2 in the trans-splicing process using in vivo and in organello approaches. When the branched intermediates formed during the splicing process were analyzed, the C residues from D6 intron domains from 3'-nad1-I4 and 3'-nad5-I2 were found changed to U, suggesting that RNA editing of these residues could be mandatory for splicing. This assumption was tested by expressing recombinant mat-r-nad1e transgenes introduced into mitochondria by electroporation. Mutation of the editing target residue dramatically affected trans-splicing. Interestingly, the exon joining efficiency was not recovered by compensatory mutations, suggesting that the role of RNA editing is not confined to the restoration of the secondary structure of domain D6 of the intron. Our results strongly support the hypothesis that RNA editing in trans-introns precedes maturation, and is required for the splicing reaction. In addition, this is the first report using an in organello approach to study the trans-splicing process, opening the way to future studies of this peculiar mechanism.