Components of a putative spliceosome involved in chloroplast RNA trans-splicing
Jessica Jacobs, Stephanie Glanz, Vera Kock, Olga Reifschneider and Ulrich Kück
Department for General and Molecular Botany, Ruhr-University Bochum, UniversiäĄtsstraße 150, 44780 Bochum, Germany,
C. reinhardtii is widely used for analysis of nucleus-encoded factors that are thought to promote the maturation of chloroplast precursor RNAs and that are presumably part of a chloroplast spliceosome [1, 2]. To elucidate the function and composition of the putative chloroplast spliceosome, we are studying the expression of the chloroplast encoded psaA gene, in particular the trans-splicing process of its precursor RNAs [3, 4]. In a forward genetic approach we used restriction enzyme-mediated integration for generation of photosynthetic mutants that are impaired in trans-splicing of psaA RNA. Here, we present three trans-splicing mutants, which are suitable for identification of novel nuclear factors involved in chloroplast trans-splicing. Genomic complementation of one of these mutants led to the identification of a gene we called RAA4, which is required for trans-splicing of the first psaA intron. It encodes a polypeptide that shares similarities with aminoacyl-tRNA synthetases. The RNA-binding properties of Raa4 were analysed by electrophoretic mobility shift assays using different organellar group II intron domains. In vivo studies using the yeast two-hybrid system led to identification of two putative interaction partners of Raa4.
[1] Perron K, Goldschmidt-Clermont M, Rochaix JD (2004) RNA 10: 704-711 [2] Glanz S, Kück U (2009) BioEssays 31:921-934 [3] Kück , Choquet Y, Schneider M, Dron M, Bennoun P (1987) EMBO J 6: 2185-2195 [4] Choquet Y, Goldschmidt-Clermont M, Girard-Bascou J, Kück U, Bennoun P, Rochaix JD (1988) Cell 52: 903-913
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