AN UNPRECEDENTED GROUP-II INTRON FROM CHLAMYDOMONAS SUBCAUDATA (CHLOROPHYCEAE) THAT HAS TWO LARGE OPEN READING FRAMES-POTENTIALLY ENCODING A REVERSE TRANSCRIPTASE-MATURASE-ENDONUCLEASE AND A NOVEL PROTEIN1

Abstract

Group-II introns are selfish ribozymes that may have given rise to nuclear mRNA introns. Approximately 1,000 of these introns—derived from organelles, bacteria, and archaea—have been defined as either having no open reading frame (ORF) or encoding a single large protein, which is nearly always a variant of a reverse transcriptase-maturase-endonuclease (RT-Mat-En). While investigating intron ribozymes in cold-tolerant Chlamydomonas spp., we discovered an unusually large (3.9 kb) group-II intron in the psbA gene of Chlamydomonas subcaudata N. Wille, Csu.psbA. Reverse transcriptase-PCR (RT-PCR) analysis showed that Csu.psbA is efficiently spliced in vivo and confirmed the predicted splice sites. The extreme size of Csu.psbA is due to two large ORFs in domain IV of the predicted secondary structure. ORF1 encodes a typical RT-Mat-En protein (70 kDa), although it has an unusual start codon (ACG). ORF2, however, encodes a potentially novel protein (44 kDa) that is predicted to have a transmembrane domain, immediately following an N-terminal thylakoid-targeting peptide, and to bind nucleic acids. BLAST analyses suggest that both ORFs are of bacterial origin and that ORF2 may have a TRKA domain. Csu.psbA is the first group-II intron reported to have two large, distinct ORFs and raises the possibility of identifying novel intron-encoded functions.