Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii

Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be at least partially recovered by increasing the Mg²⁺ concentration. The mutant constructs were transformed into chloroplasts to replace the wild-type intron; however, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4¹²⁵A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least two nuclear genes. The 7151 suppressor mutation, which defines the chloroplast-splicing suppressor (css1) gene, had no obviously altered growth phenotype with the wild-type intron, and was dominant in vegetative diploids containing the mutant intron. All three of the suppressor strains also suppressed a mutation in the P4 region of the fourth psbA intron, Cr.psbA4, indicating that these genes play a role in splicing of multiple group I introns in the chloroplast.