Principles, challenges, and advances in ribosome profiling: from bulk to low-input and single-cell analysis

Abstract Ribosome profiling has revolutionized our understanding of gene expression regulation by providing a snapshot of global translation in vivo. This powerful technique enables the investigation of the dynamics of translation initiation, elongation, and termination, and has provided insights into the regulation of protein synthesis under various conditions. Despite its widespread adoption, challenges persist in obtaining high-quality ribosome profiling data. In this review, we discuss the fundamental principles of ribosome profiling and related methodologies, including selective ribosome profiling and translation complex profiling. We also delve into quality control to assess the reliability of ribosome profiling datasets, and the efforts to improve data quality by modifying the standard procedures. Additionally, we highlight recent advancements in ribosome profiling that enable the transition from bulk to low-input and single-cell applications. Single-cell ribosome profiling has emerged as a crucial tool for exploring translation heterogeneity within specific cell populations. However, the challenges of capturing mRNAs efficiently and the sparse nature of footprint reads in single-cell ribosome profiling present ongoing obstacles. The need to refine ribosome profiling techniques remains, especially when used at the single-cell level.