The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

The mRNA maturation of the tripartite chloroplastpsaA gene from the green algaChlamydomonas reinhardtii depends on various nucleus-encoded factors that participate intrans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing thepsaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with thetrans -splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterizedtrans -splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potentialtrans -splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generatedtrans -splicing mutants. Our assay revealed that thetrans -splicing defect of mutant F19 was restored by a new factor we namedRAA8 ; molecular characterization of complemented strains verified that Raa8 participates in splicing of the firstpsaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoringpsaA mRNAtrans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifyingtrans -splicing factors.