Ultra-parallel ribosome profiling platform with RNA-dependent RNA amplification

Abstract Translation regulation plays a pivotal role in the diversification of gene expression and the response to intra- and extracellular environmental cues. Ribosome profiling (or Ribo-Seq) serves as a sensitive, quantitative, comprehensive, and data-rich technique to survey ribosome traversal across the cellular transcriptome. However, due to the intricacy of library preparation, applications to low-input and a large number of samples have presented analytic challenges. Here, we developed the semi-automated platform of Ribo-Seq and Disome-Seq, which allowed us to assess the translation status from a vast collection of samples with reduced amounts in a plate format. Through an siRNA-mediated knockdown screen for ribosome-associated proteins, this technique identified factors that (i) mediate regulation via RNA elements such as the TOP motif, (ii) assist efficient ribosome recycling, (iii) support ribosome-associated quality control (RQC), (iv) enhance translation elongation across inhibitory G-quadruplex sequences, and (v) repress mitochondrial translation. The application to human-derived samples revealed that stop codon readthrough and mitochondrial translation deficiency are associated with severe symptoms in COVID-19. Our approach provides a versatile option to investigate the translatome in a highly parallel manner. Competing Interest Statement RIKEN has submitted a patent application to the Japan Patent Office pertaining to the METHOD FOR GENERATING DNA LIBRARY USING RNA TEMPLATED RNA AMPLIFICATION aspect(s) of this work (application number PCT/JP2024/009605). S.I. is a member of the Scientific Reports editorial board. The remaining authors declare no competing interests.