Ordered processing and splicing in a polycistronic transcript in liverwort chloroplasts

From the complete sequence of the chloroplast DNA in a liverwort, Marchantia polymorpha, an unidentified open reading frame, ORF203, was found between the psbB and rps12' (trans-split) genes. ORF203 was a split gene consisting of three exons and two group II introns. Multiple transcripts for ORF203 were detected on Northern blots of the chloroplast RNA preparation. The ORF203 locus was primarily co-transcribed with the downstream genes rps12' and rpl20, and then processed into a monomeric precursor. S1 nuclease mapping gave the transcription initiation site 52 nucleotides upstream from the coding sequence of ORF203. The spliced RNA molecules were identified, as predicted, by the use of synthetic oligodeoxyribonucleotide probes specific to ligated exon sequences. The splicing reaction proceeded successively from the 5' to 3' direction. These results indicate that ordered RNA processing occurs in the chloroplast of land plants. Trans-membrane analysis by a computer indicated that ORF203 gene product could be associated with a chloroplast membrane.