SummaryThe complex maturation of the chloroplast psaA mRNA in Chlamydomonas reinhardtii requires the splicing of three separate exons in trans. At least 14 nuclear gene products and a small chloroplast RNA (tscA) seem to be involved in psaA trans-splicing. To further elucidate the mechanisms that regulate trans-splicing in C. reinhardtii, we have analyzed four different nuclear mutants; two affected in trans-splicing of the psaA exons 1 and 2 (class C) and two blocked in both psaA trans-splicing steps (class B). While mutants M18 (class C) and L118 (class B) have a mature chloroplast tscA RNA, no such transcripts could be detected in mutants TR72 (class C) and HN31 (class B). Northern hybridizations indicate that the absence of mature tscA transcripts in HN31 and TR72 results from a defect in the processing of a tscA/chlN precursor transcript. Since the tscA locus has been shown to mediate psaA exon 1 and 2 trans-splicing, the nucleus-encoded factor affected in the class C mutant TR72 may be required for tscA 3Ј end maturation and only indirectly for psaA exon 1 and 2 trans-splicing. Moreover, further genetic analysis revealed that the nuclear HN31 locus is involved in both maturation of the tscA/chlN precursor and transsplicing of the psaA exons 2 and 3.