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International audience; One sentence summary: The OPR protein MTHI1 is a major actor in the biogenesis of chloroplast ATP synthase that co-regulates the expression of AtpH and AtpI, the two subunits of the proton channel in green algae. ABSTRACT In the green alga Chlamydomonas reinhardtii, chloroplast gene expression is tightly regulated post-transcriptionally by gene-specific transacting protein factors. Here we report the identification of the octotricopeptide repeat (OPR) protein MTHI1, which is critical for the biogenesis of chloroplast ATP synthase CFo. Unlike most transacting factors characterised so far in C. reinhardtii, which control the expression of a single gene, MTHI1 targets two distinct transcripts: it is required for the accumulation and translation of atpH mRNA, encoding a subunit of the selective proton channel, but it also enhances the translation of atpI mRNA, which encodes the other subunit of the channel. MTHI1 targets the 5'UTRs of both the atpH and atpI genes. Co-immuno-precipitation and small RNA sequencing revealed that MTHI1 binds specifically a sequence highly conserved among Chlorophyceae and the Ulvale clade of Ulvophyceae at the 5'end of tri-phosphorylated atpH mRNA. A very similar sequence, located approximately 60 nt upstream of the atpI initiation codon, was also found in some Chlorophyceae and Ulvale algae species and is essential for atpI mRNA translation in C. reinhardtii. Such a dual targeted transacting factor provides a means to co-regulate the expression of the two proton hemi-channels.

Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5' exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation. RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5' end maturation is thought to be achieved by the combined action of a 5' exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5' exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5' exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5' exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

Summary as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter (ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail-anchored (TA) proteins to cytosol-exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA-homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex, which, in turn, catalyzes the import of nucleus-encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll-binding polypeptides and is not compensated by the other ARSA homolog encoded by the C. reinhardtii genome, implying a non-redundant function.