Synthetic oligonucleotide libraries reveal novel regulatory elements in Chlamydomonas chloroplast mRNAs

Gene expression in chloroplasts is highly regulated during translation by sequence and secondary-structure elements in the 5' untranslated region (UTR) of mRNAs. These chloroplast mRNA 5' UTRs interact with nuclear-encoded factors to regulate mRNA processing, stability, and translation initiation. Although several UTR elements in chloroplast mRNAs have been identified by site-directed mutagenesis, the complete set of elements required for expression of plastid mRNAs remains undefined. Here we present a synthetic biology approach using an arrayed oligonucleotide library to examine in vivo hundreds of designed variants of endogenous UTRs from Chlamydomonas reinhardtii and quantitatively identify essential regions through next-generation sequencing of thousands of mutants. We validate this strategy by characterizing the relatively well-studied 5' UTR of the psbD mRNA encoding the D2 protein in photosystem II and find that our analysis generally agrees with previous work identifying regions of importance but significantly expands and clarifies the boundaries of these regulatory regions. We then use this strategy to characterize the previously unstudied psaA 5' UTR and obtain a detailed map of regions essential for both positive and negative regulation. This analysis can be performed in a high-throughput manner relative to previous site-directed mutagenesis methods, enabling compilation of a large unbiased data set of regulatory elements of chloroplast gene expression. Finally, we create a novel synthetic UTR based on aggregate sequence analysis from the libraries and demonstrate that it significantly increases accumulation of an exogenous protein, attesting to the utility of this strategy for enhancing protein production in algal chloroplasts.