Stabilization and translation of synthetic operon-derived mRNAs in chloroplasts by sequences representing PPR protein-binding sites

The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast. Significance Statement We demonstrate that binding sites for RNA-binding proteins can be used to differentially activate translation and RNA stability of chloroplast transgenes, which is relevant for any biotechnological approach that utilizes the chloroplast as an expression platform. Intriguingly, overexpression of an sRNA sequence can lead to titration of cognate trans-acting RNA binding proteins (RBPs) followed by defects in endogenous RNA processing events at least in the case of HCF107. This suggests that RBPs can act as limiting factors of chloroplast RNA metabolism.