Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation

Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 50 untranslated regions that confer 1,000-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we determined a functional role for most alternative 50 UTR isoforms expressed in yeast, revealed a general mode of increased translation via direct binding to a core translation factor, and identified numerous translational control elements including C-rich silencers that are sufficient to repress translation both in vitro and in vivo. DART enables systematic assessment of the translational regulatory potential of 50 UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.

Automated summary

Translational control plays a crucial role in shaping the proteome under normal and pathological conditions. The DART technique allows for the systematic assessment of regulatory elements within the 50 untranslated regions, providing insights into the functional role of alternative 50 UTR isoforms and identifying translational control elements.