RNA-protein interactions in chloroplast translation

I have examined the contribution of RNA elements within the 5'-UTR of plastid mRNAs, and the protein factors that interact with these elements, to understand the mechanisms governing translation in the chloroplast of the model organism Chlamydomonas reinhardtii . Using a heterologous gfp reporter gene fused to chloroplast 5'- and 3'-UTRs, I showed that recombinant gene expression in the chloroplast is primarily dependent on the 5'-UTR. Light-regulated translation continues to operate in these chimeric mRNAs despite different levels of translational efficiency, and overall, chimeric mRNA accumulation was proportional to protein accumulation. RNA elements located in the psbA 5' -UTR include a stem-loop structure adjacent to a Shine-Dalgarno ribosome binding site. The identification and characterization of a novel RNA binding protein (RB38) that associates with the C. reinhardtii psbA 5'-UTR was shown to interact with this element in conjunction with a previously characterized poly(A) binding protein homologue (RB47, cPAB1). These proteins interact with the psbA 5'-UTR simultaneously, as well as with the coding region, to facilitate translation and potentially order the RNA such that 30S ribosomes can be recruited to the mRNA in the proper sequence context. The RNA binding activity of RB47 is modulated in vitro by a protein disulfide isomerase (RB60, cPDI), and the cloning of an oxidoreductase (Tba1) has identified another candidate involved in this redox-regulated cascade of psbA mRNA translation. The development of an RNAi-based strategy, shown to silence GFP expression, was successfully employed to identify a C. reinhardtii mutant strain deficient in RB60 protein accumulation. The loss of RB60 correlates with a slight decrease in RB47 psbA mRNA binding activity, yet photosynthesis in general, and D1 protein synthesis specifically, was not affected in this mutant. The lack of phenotype in an RB60 knockdown strain was subsequently shown to be due to a functionally redundant PDI activity in the chloroplast. This newly identified PDI (CrPDI) is a chloroplast soluble protein that co-purifies with the psbA ribonucleoprotein complex, suggesting that the fourth member of the psbA-associated complex, RB55, is a second protein disulfide isomerase. (Abstract shortened by UMI.)...