Ribosome stalling facilitates chloroplast targeting of nuclear-encoded proteins

Abstract Ribosomes translate mRNAs with variable elongation rates and frequently undergo transient stalling. Although ribosome stalling is known to regulate protein quality control and translational dynamics, its transcriptome-wide landscape and biological significance in plants remain largely unexplored. Here, using disome profiling in Arabidopsis thaliana , we generated a high-resolution map of ribosome stalling sites across the transcriptome and uncovered a marked enrichment on mRNAs encoding chloroplast-targeted proteins, particularly those involved in photosynthesis. We further found that ribosome stalling preferentially occurs when transit peptides emerge from the ribosome exit tunnel. Functional assays demonstrated that deletion of the stalling region significantly reduces chloroplast targeting efficiency. Together, our findings identify ribosome stalling as a regulatory layer that promotes efficient targeting of nuclear-encoded proteins to chloroplasts.