Towards unravelling the chloroplast spliceosome of Chlamydomonas reinhardtii

Stephanie Glanz and Ulrich Kück

Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany

The unicellular green alga C. reinhardtii is widely used for analyzing nuclear-encoded factors that are thought to promote the maturation of chloroplast precursor RNAs and are presumably part of a postulated chloroplast spliceosome. As an example, the expression of the psaA gene, in particular the trans-splicing process of its precursor RNAs is studied. Here, we present two approaches that enable the isolation of novel nuclear-encoded factors. In a forward genetic approach, we used restriction enzyme-mediated integration to generate novel trans-splicing mutants. Genomic complementation of one of these mutants identified a region of the nuclear DNA which is deleted in the mutant strain compared to the wild type. This locus encodes a polypeptide with similarities to aminoacyl-tRNA synthetases. In the second approach, the yeast three-hybrid system was used to isolate the chloroplast nucleosome assembly protein-like cNAPL. The RNA-binding property was demonstrated by electrophoretic mobility shift assays using different organellar group II intron domains. The chloroplast localization of cNAPL was determined by laser scanning confocal fluorescence microscopy. Phylogenetic analysis showed that no homologues of cNAPL and its related counterparts are present in prokaryotic genomes. The availability of several trans-splicing mutants from C. reinhardtii and in vitro studies with RNA-binding proteins will further identify components, which are most probably part of the chloroplast spliceosome.

e-mail address of presenting author: Stephanie.Glanz@rub.de