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...

In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaAi2, splice in trans, and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatographymass spectrometry (LC-MS), size exclusion chromatography, and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein apparatus participating in psaA mRNA splicing. This supercomplex contains two subcomplexes (I and II) that are responsible for trans-splicing of either psaA-i1 or psaA-i2. We further demonstrate that both subcomplexes are associated with intron RNA, which is a prerequisite for the correct assembly of subcomplex I. This study contributes further to our view of how the eukaryotic nuclear spliceosome evolved after bacterial endosymbiosis through fragmentation of self-splicing group II introns into a dynamic, protein-rich RNP machinery.The spliceosome is a dynamic RNP machinery that participates in the excision of mRNA introns in eukaryotes. This machinery consists of five small nuclear RNAs (snRNAs; U1, U2, U4, U5, and U6) and a large number of spliceosomal proteins (1, 2). It is generally accepted that spliceosomal snRNAs were derived from ancient group II introns, which were introduced into the eukaryotic cell after endosymbiosis (3, 4). Group II introns occur frequently in bacteria and organelles of fungi, algae, and higher plants but are rare in archaea and absent from nuclear genomes. Comparable with spliceosomal introns, the splicing reaction includes two transesterification reactions, yielding spliced exons and an excised intron lariat RNA.Despite the lack of significant sequence similarities, group II introns share a common secondary structure with six helical domains (D1-D6) surrounding a central wheel. During intron excision, these domains take over specific functions, and remarkably, they show plenty of functional and structural similarities to spliceosomal snRNAs (5-7). These similarities led to the assumption that during evolution of the nuclear splicing apparatus, group II introns fragmented and degenerated to spliceosomal snRNAs (3,8,9).In contrast to bacterial group II introns, most organelle group II introns display variant forms of degeneration and fragmentation (9). For example, many of the plant group II introns have mispaired domain structures (10, 11). Moreover, group II introns are able to split into autonomous fragments due to rearrangements of organelle genomes (12, 13). Consequently, they are transcribed independently, and association of precursor RNAs by base pairing generates a catalytically active group II intron structure that is finally processed by trans-splicing. Such degenerated and fragmented group II introns along with the interplay of discrete RNAs during the splicing reaction serve to demonstrate how snRNAs...

SUMMARYSplicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity.