Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features. The splicing mechanism of group II introns and the secondary structures of their presumed active sites were used as early arguments for the hypothesis that this class of introns represents the ancestors of eukaryotic spliceosomal introns (1, 2). It was further assumed that group II introns invaded the eukaryotic nucleus and subsequently proliferated at various genomic sites, leading to the degeneration of the catalytic intron structure into small nuclear RNAs (snRNAs).1 This assumption was supported by the observation of naturally occurring variants of group II introns that are split into two or more pieces (3), reminiscent of eukaryotic spliceosomal RNA (1). Group II intron RNAs are characterized by six conserved domains, and tertiary interactions among these domains generate the compact native and catalytic complex. Some of these group II intron domains have been shown to act in trans on the splicing of other introns that lack the corresponding domain (4). In vivo, various RNA-binding proteins promote the formation of catalytically active intron RNA. In contrast to the nuclear spliceosome, which acts generally on a broad range of nuclear-encoded pre-mRNAs, proteins involved in organellar intron splicing seem to more efficiently stabilize the active three-dimensional RNA structure in vivo. Several splicing factors in higher plants, such as the chloroplast RNA-splicing and ribosome maturation (CRM) domain protein CRS1, as well as the pentatricopeptide repeat proteins OTP51 and PPR4, have been reported to be involved in the splicing of single transcripts (5, 6). Nonetheless, there are splicing factors that carry out functions on a broad range of transcripts, including CRS2 and its associated proteins CAF1 and CAF2, and WTF1, a splicing factor containing a plant organelle RNA-recognition domain (5, 6). Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5, 7). I...

SUMMARYDuring trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P 700 core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in transsplicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNAbinding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.