How does ribosome profiling work and what insights does it provide?

Ribosome profiling (Ribo-seq) provides genome-wide, nucleotide-resolution snapshots of translation. The technique involves arresting translation, RNase digestion to retain only ribosome-protected fragments (RPFs, ~28-32nt), ribosome isolation, RNA extraction, library preparation, and deep sequencing. Analysis reveals: actively translated ORFs (including uORFs, overlapping ORFs, novel peptides), translation efficiency (Ribo-seq/RNA-seq ratio), ribosome pausing sites, reading frame, and translation dynamics. Challenges include library preparation biases, distinguishing true signal from contamination, and statistical analysis of differential translation. Applications include identifying functional micropeptides, understanding translational regulation, and drug mechanism studies (cycloheximide vs other inhibitors reveal different aspects).