Ribosoomprofilering

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.