Correct in vivo RNA splicing of a mitochondrial intron in algal chloroplasts.

Abstract

The self-splicing group II intron (rl1) from Scenedesmus obliquus mitochondria together with its 6 bp intron binding site (IBS1) were inserted in the correct and inverse orientation into the chloroplast tscA gene from C.reinhardtii. Precursor RNA derived from the chimeric tscA-rl1 gene can be used to demonstrate in vitro self-splicing of the rl1 intron RNA. Using the particle bombardment technique, the tscA-rl1 construct was transferred into the chloroplast of the unicellular alga Chlamydomonas reinhardtii. We recovered transformants which contain the chimeric tscA-rl1 gene as shown by Southern analysis. Hybridization and PCR analysis of transcripts confirmed that the heterologous intron is correctly spliced in vivo. From sequencing of cDNA clones we conclude that the IBS1 sequence is sufficient for correct splicing of the mitochondrial intron in C. reinhardtii chloroplasts. Using specific probes, we demonstrate by Northern hybridization that the mature RNA, as well as an intron-3' exon intermediate, accumulate in transformants containing the rl1 intron, correctly inserted into the tscA gene. As expected, no RNA splicing at all was observed when the intron had an inverted orientation within the tscA gene. In addition, a mutated intron RNA with an altered 3' terminal nucleotide was tested in vivo. In contrast to similar mutants examined in vitro, this mutated RNA shows accumulated intron and intron-3' exon intermediates, but no ligated exons at all. Our approach should prove useful for elucidating nucleotide residues involved in splicing of organelle introns in vivo.