Functional analysis of Raa4, a chloroplast splicing factor from Chlamydomonas reinhardtii
Vera Kock, Stephanie Glanz, Jessica Jacobs and Ulrich Kück
Department for General and Molecular Botany, Ruhr-University Bochum, Universitässtraße 150, 44780 Bochum, Germany,
The psaA gene of Chlamydomonas reinhardtii comprises three independently transcribed exons, which are flanked by consensus sequences of group IIB introns [1, 2]. Trans-splicing of the psaA pre-mRNA requires at least 14 nucleus-encoded factors and a small chloroplast-encoded RNA (tscA) [3]. In a forward genetic approach we used restriction enzyme-mediated integration to generate novel trans-splicing mutants and rescued one of these mutants by genomic complementation. The affected gene is called Raa4 and does not share significant sequences similarities with any other protein except for a small domain present in aminoacyl-t RNA synthetases. For the detection of putative Raa4 interaction partners we applied yeast two-hybrid studies using the MatchmakerTM system from Clontech. We tested 1,4 x 109 interactions with a mating efficiency of 37% and identified 98 clones with significant reporter gene activity. One of these putative interaction partners called Rab1 (Raa4-binding 1 protein) contains a putative RNA-binding site and a chloroplast transit peptide.

[1] Glanz S,Kück U (2009) BioEssays 31:921-934
[2] Kück U, Choquet Y, Schneider M, Dron M, Bennoun P (1987) EMBO J 6: 2185-2195
[3] Choquet Y, Goldschmidt-Clermont M, Girard-Bascou J, Kück U, Bennoun P, Rochaix JD (1988) Cell 52: 903-913
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