5.15
Ribosome Profiling
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequence…
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.
View the full transcript and gain access to JoVE Core videos
Q1: What is ribosome profiling and what does it measure?
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.
Q2: How does ribosome profiling isolate and protect mRNA fragments?
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.
Q3: What role do open reading frames play in ribosome profiling 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.
Q4: How is ribosomal RNA contamination addressed in ribosome profiling?
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.
Q5: What technical challenges does ribosome profiling face?
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.
Q6: How does ribosome profiling convert mRNA to DNA for sequencing?
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.
Q7: What applications does ribosome profiling have in studying gene expression?
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.