5′ and 3′ ends of chloroplast transcripts can both be stabilised by protein ‘caps': a new model for polycistronic RNA maturation

Chloroplast gene expression relies on nucleus-encoded factors acting posttranscriptionally in a gene-specific manner. Among those, RNA stability factors bind to the 50UTR of their target RNAs to protect them from 50-30 exonucleases. By contrast, little was know, up to now, on the molecular events involved in the complex processing of chloroplast polycistronic transcripts. In this issue of The EMBO Journal, Pfalz et al convincingly demonstrate that PPR10, a maize PPR protein, binds a conserved sequence in the intergenic regions of two distinct polycistronic transcripts. Once bound, PPR10 defines the termini of the processed RNAs issued from these polycistronic precursors by impeding the progression of exonucleases acting from both the 50 and 30 directions. Other PPR proteins likely acting similarly, Barkan and co-workers (Pfalz et al, 2009) propose a new and stimulating model for the maturation of chloroplast transcripts that would involve poorly specific endonucleases and secondary structures or bound proteins that protect transcripts from 50-30 or 3050 exoribonucleases. Chloroplasts evolved from free-living cyanobacteria captured by a primitive eukaryotic cell. They have retained from their ancestor a prokaryotic-like gene expression machinery and polycistronic transcription units. These latter, however, do not merely correspond to bacterial operons as their expression is not controlled by specific transcriptional repressors/activators. Furthermore, most polycistronic transcripts comprise genes contributing different functions and are often trimmed to monocistronic RNAs. After endosymbiosis, most genes of the endosymbiont, including a subset of those encoding subunits of the photosynthetic apparatus, were transferred to the nucleus of the host. This massive gene transfer, together with the differentiation in plants of various types of plastids, necessitated new strategies to coordinate the expression of the nuclear and organelle genomes. As a result, the regulation of organelle genes expression now differs widely from that prevailing in cyanobacteria: transcriptional regulations only play a limited role in chloroplasts, where gene expression is mainly controlled at the posttranscriptional level. Posttranscriptional steps of organelle genes expression include cisand transsplicing, editing, cleavage between the coding regions by endonucleases, processing of RNA 50and 30-ends by exonucleases and translational activation. These latter RNA maturation events generate for a given polycistronic unit a complex pattern of monoand oligo-cistronic RNAs. Each of these posttranscriptional steps is tightly controlled by trans-acting factors of nuclear origin (reviewed in Barkan and Goldschmidt-Clermont, 2000). Strikingly, most of these factors are gene specific, one factor being required for the expression of one, or a few, organelle mRNA(s). Altogether, several hundred nucleus-encoded factors should be required for the proper expression of the organelle genome. The PPR protein family, named from the repetition of a 35 residue degenerate motif (Small and Peeters, 2000), is highly represented among these trans-acting factors. PPR proteins are found in all eukaryotes but this family is greatly expanded in land plants, with 4450 members in Arabidopsis or rice. Most PPR proteins are targeted to organelles, where they interact specifically with one or a few target mRNA(s) to assist the posttranscriptional steps of gene expression (reviewed in Schmitz-Linneweber and Small, 2008). Although several PPR proteins have been characterised, their mode of action is still poorly understood. Up to now, chloroplast RNA metabolism was thought to result from the interplay between distinct 50-30 and 30-50 exonucleases and sequence-specific endonucleases (reviewed in Bollenbach et al, 2004). Sequence-specific endonucleases would cleave the polycistronic transcripts within intergenic regions. Gene-specific trans-acting factors encoded in the nucleus would bind the 50 UTR of their chloroplast mRNA targets and protect them against 50-30 exonucleases, whose role in chloroplast mRNA decay pathways is well established (Drager et al, 1998). Chloroplast transcripts would be further stabilised by stable stem-loops structures at their 30 ends, protecting them, in a rather unspecific way, from 30-50 exonucleotidic degradation. This model, however, failed to account for several puzzling observations: (1) some chloroplast transcripts lack stable stem-loop structures at their 30ends; (2) in several instances, as described in this issue by Barkan and co-workers for the maize transcription units atpI-atpH, psaJ-rpl33, psbH-petB and petB-petD, the 50 end of the downstream transcript in a polycistronic unit overlaps by about 20–30 nts the 30 end of the upstream transcript, in a The EMBO Journal (2009) 28, 1989–1990 | & 2009 European Molecular Biology Organization | Some Rights Reserved 0261-4189/09 www.embojournal.org