16.17: Ribosome Profiling
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps identify translated open reading frames and discover new translated products, including short peptides and isoforms of characterized proteins with unknown functions. Ribosome profiling also aids in identifying several mRNAs in the cell that are not translated until they receive an external signal.
Technical challenges of Ribosome Profiling
Ribosome profiling faces many technical challenges, such as the requirement for large amounts of samples, dependability on timely inhibiting of translation, and RNA contamination.
The flash-freezing technique can overcome the challenge of rapid translation inhibition. It helps to efficiently capture the ribosome distribution within cells compared to translation elongation inhibitors, such as cycloheximide.
Ribosomal RNA (rRNA) that binds to the mRNA is generally removed during ribosome profiling. However, sometimes a few rRNA contaminants are still observed. Researchers use the duplex-specific nuclease (DSN) enzyme to reduce rRNA contamination, isolated from the hepatopancreas of the Kamchatka crab, that cleaves dsDNA and DNA-RNA hybrids. This method is often also used to normalize cDNA libraries before next-generation sequencing and the exhaustion of rRNA from RNA-seq libraries.
Another limitation of ribosome profiling is in analyzing data, which requires the right expertise in bioinformatics. An R package riboSeqR is used to overcome this issue, which provides methods for resolving ribosomal profiling data for multiple samples.