The Chloroplast Trans-Splicing RNA-Protein Supercomplex from the Green Alga Chlamydomonas reinhardtii

Abstract

In eukaryotes, RNA trans-splicing is a significant RNA modification process for the end-to-end ligation of exons from separately transcribed primary transcripts to generate mature mRNA. So far, three different categories of RNA trans-splicing have been found in organisms within a diverse range. Here, we review trans-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. We discuss the origin of intronic sequences and the evolutionary relationship between chloroplast ribonucleoprotein complexes and the nuclear spliceosome. Finally, we focus on the ribonucleoprotein supercomplex involved in trans-splicing of chloroplast group II introns from the green alga Chlamydomonas reinhardtii. This complex has been well characterized genetically and biochemically, resulting in a detailed picture of the chloroplast ribonucleoprotein supercomplex. This information contributes substantially to our understanding of the function of RNA-processing machineries and might provide a blueprint for other splicing complexes involved in trans- as well as cis-splicing of organellar intron RNAs.

Keywords: Chlamydomonas reinhardtii; chloroplast; group II intron; ribonucleoprotein complex; trans-splicing.

Figure 1

The introns-late theory explains the evolution of spliceosomal introns. The endosymbiosis of a group II intron-rich α-proteobacteria into the archaeal intronless host was followed by the invasion of the host genome by mobile group II introns. The resulting discontinuous genes provoked a strong selective pressure toward evolving intron removal. This included the degradation of group II intron RNA into mRNA introns and small nucleolar RNAs (snRNAs). Separation of the inefficient splicing reaction from translation was achieved by developing a nuclear envelope.

Figure 2

Secondary structure of psaA-i1 and psaA-i2 intron RNAs from C. reinhardtii. The three exons of the psaA gene and tscA locus of C. reinhardtii are transcribed independently. Two group II introns are formed at the exon boundaries of psaA by RNA base pairing. The mature psaA mRNA is generated by two trans-splicing reactions. Both group II introns have a characteristic group II intron structure, comprising six helical domains (DI-DVI), which surround a central core. psaA-i1 is a tripartite intron, whereas psaA-i2 is a dipartite intron. Fragmentation sites are indicated by arrows. Abbreviations: EBS/IBS, exon-/intron-binding sites.

Figure 3

RNPs of group II and spliceosomal introns. (A) The majority of bacterial group II introns form an RNP with the intron-encoded maturase during the splicing reactions [14]. (B) In organelles, group II introns are degenerated and RNPs comprise at least five splicing factors [20]. The maturase is either encoded by an organellar group II intron (matR, matK) sequence or is nucleus-encoded (nMat1–4; [28]). (C) Fragmented group II introns of C. reinhardtii depend on complex RNPs comprising up to ten splicing factors and the precursor RNAs. A maturase homolog has not been identified yet. (D) The five trans-acting snRNAs of the nuclear spliceosome probably originated from fragmentation of group II intron sequences. snRNAs associate with a large number of protein factors to form a complex with snRNPs and function in splicing of nuclear mRNA introns (reviewed in [4]). Homologies to group II intron maturases were shown for the splicing factor Prp8 [60]. Abbreviations: M, maturase; Mt, mitochondrial; cp, chloroplast.

Figure 4

Model of subcomplex I and II involved in psaA trans-splicing. Comparative TAP analyses revealed that subcomplex I comprises at least ten core subunits. Five of these were previously characterized as splicing factors, namely Raa1, Raa3, Raa4, Raa8, and Rat2. Subcomplex I is an RNP and copurifies with psaA exon 1 precursor and tscA RNA [123]. Subcomplex II participates in the second psaA splicing reaction and comprises at least seven subunits [143]. These were identified by TAP technology, using Raa2 and Raa7 as baits. Copurification of psaA exon 2 and exon 3 precursors indicate that the membrane-associated (thylakoid membrane at the top) subcomplex II is able to form an RNP. Bait proteins used in this study for TAP are denoted with an anchor. Proteins involved in both psaA splicing reactions are highlighted in yellow, psaA-i1 splicing factors are colored in red, and psaA-i2 splicing factors are colored in orange. Uncharacterized proteins are denoted by their molecular mass in kDa. Subcomplex I: 71 kDa = Cre17.g724450; 109 kDa = Cre12.g533351; 109 kDa OPR = Cre01.g001501; 113 kDa = Cre08.g373878; 139 kDa = Cre11.g467652. Subcomplex II: 52 kDa = Cre03.g179000; 58 kDa = Cre17.g728850; 67 kDa = Cre02.g073200 (OPR, octatricopeptide repeat).