Molecular identification and function of cis- and trans-acting determinants for petA transcript stability in Chlamydomonas reinhardtii chloroplasts.

In organelles, the posttranscriptional steps of gene expression are tightly controlled by nucleus-encoded factors, most often acting in a gene-specific manner. Despite the molecular identification of a growing number of factors, their mode of action remains largely unknown. In the green alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene, which codes for cytochrome f, depends on two specific nucleus-encoded factors. MCA1 controls the accumulation of the transcript, while TCA1 is required for its translation. We report here the cloning of MCA1, the first pentatricopeptide repeat protein functionally identified in this organism. By chloroplast transformation with modified petA genes, we investigated the function of MCA1 in vivo. We demonstrate that MCA1 acts on the very first 21 nucleotides of the petA 5 untranslated region to protect the whole transcript from 533 degradation but does not process the 5 end of the petA mRNA. MCA1 and TCA1 recognize adjacent targets and probably interact together for efficient expression of petA mRNA. MCA1, although not strictly required for translation, shows features of a translational enhancer, presumably by assisting the binding of TCA1 to its own target. Conversely, TCA1 participates to the full stabilization of the transcript through its interaction with MCA1. Organelle genomes have retained a limited set of genes from their prokaryotic ancestor, the expression of which is tightly controlled by the nucleus. Within organelles, mRNAs may undergo cis or trans splicing, editing, endo- and exonucleolytic cleavage, and 5- and 3-end processing. Their stabilization, translation, and degradation are highly regulated (reviewed in references 3, 5, 23, 26, and 63). Each of these posttranscriptional steps depends on nucleus-encoded factors. Strikingly, most are gene specific, one factor being required for the expression of one or a few organelle mRNAs. Altogether, several hundreds of nucleus-encoded factors may be required for the proper expression of the organelle genome (3, 65). The green alga Chlamydomonas reinhardtii and the yeast Saccharomyces cerevisiae have been instrumental in the identification of these factors. Genetic analyses of nuclear mutants, defective for the expression of single organelle genes, have emphasized the existence of two major classes of trans-acting factors. Some are required for the proper maturation and stabilization of specific organellar transcripts (reviewed in ref