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Ribosome profiling, also called ribo-seq, is a deep sequencing technique that maps ribosome positions on mRNA to identify actively translated genes. It produces a snapshot of the translation landscape in a cell at any given moment by selectively sequencing mRNAs protected by ribosomes, revealing which genes are being translated into proteins.

Cells are lysed to access internal molecules, then the lysate is treated with RNases that cleave unprotected mRNAs. Ribosomes shield their bound mRNA from cleavage, leaving only ribosome-protected fragments. These protected fragments are then separated from cleaved fragments using a sucrose gradient for further analysis.

Ribosome profiling identifies open reading frames (ORFs), which are DNA regions between start and stop codons that can be translated into proteins. Discovering unrecognized ORFs helps identify new genes and novel proteins. In experimental studies, ribosome profiling can reveal previously unknown translated regions upregulated by specific cellular stimuli.

Ribosomal RNA (rRNA) that binds to mRNA is typically removed during ribosome profiling. However, rRNA contaminants sometimes persist. Researchers use the duplex-specific nuclease (DSN) enzyme, isolated from the Kamchatka crab hepatopancreas, which cleaves dsDNA and DNA-RNA hybrids to reduce rRNA contamination in samples.

Ribosome profiling requires large sample amounts, timely translation inhibition, and managing RNA contamination. Flash-freezing efficiently captures ribosome distribution compared to elongation inhibitors like cycloheximide. Data analysis also demands bioinformatics expertise; the riboSeqR R package helps overcome this limitation by providing methods for resolving profiling data across multiple samples.

After ribosomes are removed from protected mRNA fragments, the RNA is converted into DNA using reverse transcriptase through RT-PCR. The resulting DNA is then sequenced and mapped onto a reference genome to determine the exact ribosome location along each mRNA molecule.

Ribosome profiling enables in vivo monitoring of translation in specific organs or tissues and quantifies new protein synthesis levels. It discovers translated products including short peptides and protein isoforms with unknown functions. The technique also identifies mRNAs that remain untranslated until receiving external signals, revealing dynamic translation regulation.