Ribosome profiling, or Ribo-seq, provides a snapshot of protein synthesis within a cell. The technique identifies which messenger RNA (mRNA) strands are being actively translated by ribosomes. It determines both the quantity of ribosomes on a gene and their precise positions, revealing which genes are producing proteins, not just which are being transcribed.
The core principle is that a ribosome physically shields the small portion of mRNA it is translating. By capturing and sequencing these protected fragments, researchers create a high-resolution map of ribosome activity. This information reveals the rate at which genes are translated, a process that static measurements of mRNA levels alone cannot provide.
Cell Lysis and Ribosome Arrest
The first step is to halt all translational activity and gently break open the cells to preserve a snapshot of protein synthesis. This is achieved by treating cells with a translation inhibitor, such as cycloheximide, which freezes elongating ribosomes on their mRNA templates. This process ensures the captured ribosome positions reflect their in-vivo locations.
Once translation is arrested, the cells are broken open through lysis. The goal is to release the cellular contents, including complexes of mRNA with multiple ribosomes (polysomes), while keeping these structures intact. This is done using gentle detergents and mechanical disruption in ice-cold buffers to preserve the ribosome-mRNA complexes.
Nuclease Digestion and Monosome Isolation
With the cellular contents released into a lysate, the next objective is to process the polysomes. The lysate is treated with a ribonuclease (RNase) that digests any mRNA not physically shielded by a ribosome. This enzyme cleaves all exposed single-stranded RNA.
The result is a mixture of individual ribosomes, each protecting an mRNA fragment of approximately 28 to 30 nucleotides. These ribosome-mRNA complexes are called monosomes, and the protected RNA is the “footprint.” These monosomes must be separated from other cellular components like unincorporated amino acids and tRNAs.
This purification is achieved through sucrose density gradient ultracentrifugation. The lysate is layered onto a sucrose gradient and spun at high speeds, separating molecules by size and shape. The heavier monosomes form a distinct band that can be collected, yielding a purified sample of ribosome-protected fragments.
Sequencing Library Preparation
After isolating the monosomes, the ribosome-protected fragments (RPFs) are extracted and prepared for sequencing. This process converts the RNA footprints into a library that can be read by a next-generation sequencing machine. The first step is extracting the RPFs from the ribosome complexes, using a denaturing gel to select for fragments between 24 and 36 nucleotides.
A synthetic adapter is attached to the 3′ end of each RNA fragment. This adapter serves as a starting point for reverse transcription, where an enzyme converts the RNA fragments into more stable complementary DNA (cDNA). The resulting single-stranded cDNA is then circularized to place the fragment between the necessary sequencing sequences.
The final step is amplification through a polymerase chain reaction (PCR), which makes millions of copies of each cDNA molecule. The amplification primers also add the remaining sequences required for the library molecules to bind to the sequencer’s flow cell. The final product is a pool of DNA molecules ready for high-throughput sequencing.
Data Processing and Analysis
The final stage is computational, transforming raw sequencing data into biological insights. The output from the sequencer is a massive data file containing millions of short DNA sequences, or reads. The first processing step involves trimming the adapter sequences added during library preparation and filtering to remove low-quality reads or those outside the expected length of 25 to 35 nucleotides.
The high-quality reads are then aligned to a reference genome or transcriptome to identify the gene from which each footprint originated. An analytical step is determining the P-site (peptidyl site) offset for the reads. This calculation infers the precise codon the ribosome was positioned over by using the footprint’s length and mapped location. This provides single-codon resolution, a level of precision that is a primary feature of the technique.
With all footprints mapped, researchers can analyze ribosome density, which is the number of reads aligned to a particular gene. By comparing this to the gene’s mRNA abundance from a parallel RNA-seq experiment, one can calculate a measure of translation efficiency. The data can reveal where translation starts, identify ribosome pausing events that may indicate regulatory control, and discover previously unknown translated regions of the genome. The final output is visualized as a plot showing ribosome occupancy along a gene, providing a clear picture of its translation dynamics.