Uncovering the role of the PPR protein PHOTOSYSTEM ONE BIOGENESIS FACTOR6 in splicing chloroplast group II introns

Nucleus-encoded proteins are involved in intron splicing in plant chloroplasts. Although many splicing factors were discovered, the mechanisms for chloroplast intron splicing remain unknown. Here, we identified a P-class pentatricopeptide repeat (PPR) protein, PHOTOSYSTEM ONE BIOGENESIS FACTOR6 (PBF6), that is essential for the accumulation of photosystem I complex. PBF6 bound to ycf3 intron 1 and clpP1 intron 2, and the introns of petB, ndhA, and ndhB transcripts, and was required for their splicing. Truncated PBF6 containing the 10 N-terminal PPR motifs bound to specific sequences to ycf3 intron 1, clpP1 intron 2, and to the introns of petB, ndhA, and ndhB. PBF6 formed two multi-subunit splicing complexes with other known splicing factors from the CRM, peptidyl-tRNA hydrolase, RNase III, PORR, APO, DEAD-box RNA helicase, and mTERF families. PBF6-containing complex I was about 600 kDa, comprising nine known splicing factors: CAF1, CAF2, CRS2, CFM3a, RNC1, WTF1, APO2, RH3, and mTERF2. PBF6-containing complex II was about 300 kDa and contained three known splicing factors: CAF1, CAF2, and CFM2. Furthermore, two known PPR-type splicing factors, PBF2 and ECD2, both of which are required for splicing the ycf3 intron 1, also formed two multi-subunit splicing complexes with other known splicing factors. Importantly, these three PPR-type splicing factors formed their splicing complexes independently. Our data suggest that a PPR-type splicing factor forms splicing complexes with other known splicing factors under transient expression conditions to facilitate intron splicing and that several PPR-type splicing factors work together to promote the splicing of the same intron through forming respective splicing complexes.