Spleißosom-ähnliche Komplexe in Chloroplasten der Grünalge Chlamydomonas reinhardtii\textit {Chlamydomonas reinhardtii}

Bei der Reifung der $\it psaA$-mRNA in Chloroplasten der Grünalge $\textit{Chlamydomonas reinhardtii}$ werden zwei diskontinuierliche Gruppe II-Intronen durch zwei $\it trans$-Spleißreaktionen entfernt. Gruppe II-Intronen gelten als Vorläufer von nukleären mRNA-Intronen. Der Spleißprozess der $\it psaA$-mRNA ist abhängig von zahlreichen nukleär-kodierten Proteinen. Um die Ausbildung eines putativen plastidären Spleißosoms zu untersuchen, wurden in dieser Arbeit umfangreiche Tandem-Affinitätsaufreinigungen, massenspektrometrische Analysen und biochemische Methoden durchgeführt. Es konnte gezeigt werden, dass die $\it psaA$-mRNA-Spleißfaktoren mit zahlreichen weiteren Proteinen zwei hochmolekulare Ribonukleoprotein-Komplexe im Verlauf des $\it trans$-Spleißens ausbilden. Darüber hinaus konnten Hinweise für die Ausbildung eines Suprakomplexes, der beide Spleißreaktionen verbindet, gefunden werden. Der plastidäre Spleißapparat der Grünalge weist damit viele Ähnlichkeiten mit der Spleißmaschinerie nukleärer mRNA-Intronen auf

Identification of a chloroplast ribonucleoprotein complex containing trans-splicing factors, intron RNA, and novel components

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. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns.14 page(s

A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii

Splicing 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