P‐class pentatricopeptide repeat protein PTSF1 is required for splicing of the plastid pre‐tRNAIle in Physcomitrella patens

Pentatricopeptide repeat (PPR) proteins are widely distributed in eukaryotes and are mostly localized in mitochondria or plastids. PPR proteins play essential roles in various RNA processing steps in organelles; however, the function of the majority of PPR proteins remains unknown. To examine the function of plastid PPR proteins, PpPPR_4 gene knock-out mutants were characterized in Physcomitrella patens. The knock-out mosses displayed severe growth retardation and reduced effective quantum yield of photosystem II. Immunoblot analysis showed that knock-out of PpPPR_4 resulted in a strongly reduced level of plastid-encoded proteins, such as photosystem II reaction center protein D1, the beta subunit of ATP synthase, and the stromal enzyme, Rubisco. To further investigate whether knock-out of the PpPPR_4 gene affects plastid gene expression, we analyzed steady-state transcript levels of protein-and rRNA-coding genes by quantitative RT-PCR. This analysis showed that the level of many protein-coding transcripts increased in the mutants. In contrast, splicing of a spacer tRNA(Ile) precursor encoded by the rrn operon was specifically impaired in the mutants, whereas the accumulation of other plastid tRNAs and rRNAs was not largely affected. Thus, the defect in tRNA(Ile) splicing leads to a considerable reduction of mature tRNA(Ile), which may be accountable for the reduced protein level. An RNA mobility shift assay showed that the recombinant PpPPR_4 bound preferentially to domain III of the tRNA(Ile) group-II intron. These results provide evidence that PpPPR_4 functions in RNA splicing of the tRNA(Ile) intron, and hence PpPPR_4 was named plastid tRNA splicing factor 1 (PTSF1).