16.15: 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.

Identifying regions in a genome that are transcribed and translated into proteins is a vital step in understanding a genome and its expression.

This is achieved with a technique called ribosome profiling, also referred to as ribo-seq. It maps the positions of ribosomes on mRNA and identifies mRNAs that are being actively translated into proteins.

To isolate the RNA, cells must first be lysed to access the molecules inside them. The lysate is then treated with RNases. These enzymes cleave the mRNAs that are not covered with ribosomes, leaving only the protected mRNA fragments.

The ribosome protected fragments are then separated from the unprotected, cleaved fragments using a sucrose gradient.

The ribosomes are then removed from the mRNA fragments, and the RNA are converted into DNA by RT-PCR, using the enzyme reverse transcriptase.

Next, the DNA is sequenced and mapped on the reference genome. This determines the exact location of the ribosome along each mRNA.

Ribosome profiling can also help to identify unrecognized open reading frames or ORFs. An ORF is a region of DNA between a start codon and a stop codon that can be translated into protein. Finding ORFs can be helpful in the identification of new genes.

Consider the study of gene expression patterns in a mammalian cell line. The experimental procedure involves exposing the cells to stimuli that may turn on gene expression and initiate mRNA synthesis.

The mRNAs that are being actively translated are identified using ribosome profiling.

This experiment reveals that gene B is being translated while genes A and C are not.

It also shows an additional small region of the genome around a hundred nucleotides long is being actively translated.

This unrecognized region is an open reading frame that may code for a novel protein that is upregulated by the experimental stimuli.