Genome-wide analyses of translational dynamics in chloroplasts

The translation of chloroplast mRNAs has long been known to be subject to regulation by developmental, environmental and physiological cues. However, progress in recognizing examples of translational regulation, identifying translational regulators, and dissecting mechanisms of translational modulation has been limited by the assays that have been available to monitor ribosome behavior in vivo: the traditional assays (pulse-labeling, polysome, and reporter gene approaches) are labor intensive, have limited sensitivity and/or resolution, and are not suited to genome-wide explorations. We are using genome-wide ribosome profiling methods that provide a quantitative and high resolution readout of ribosome positions in vivo to (i) identify nucleus-encoded proteins that are required for the translation of specific chloroplast mRNAs; (ii) analyze the impact of various light regimes on chloroplast ribosome behavior; (iii) describe the translational dynamics of organellar mRNAs during the differentiation of photosynthetic leaf cells; and (iv) elucidate mechanisms involved in the co-translational targeting of proteins to the chloroplast thylakoid membrane. Examples of insights obtained in each of these areas will be discussed.