In the past decade, second-generation sequencing has been rapidly developed and widely used in the studies of genome, transcriptome, and epigenetics of many species. It also acts as an important technological support for pharmacy, diagnosis, and many other fields. In recent years, second-generation sequencing has been increasingly used to monitor protein translation. Combining ribosome profiling and deep sequencing, researchers can monitor protein translation precisely at the genomic level.
Ribosome profiling is also referred to as ribosome footprinting or Ribo-Seq. It is a transcriptomic research technique that measures translation comprehensively and quantitatively by identifying the precise positions of ribosomes to determine which mRNAs are being actively translated. This allows for the detection of the active ribosomes in a cell at a particular moment and enables researchers to identify the location of the translation initiation sites, the complement of translated ORFs, the specific locations of ribosomes on the mRNA, and the translation rate of ribosomes. There could be a distinct difference in protein translation rates while mRNAs are transcribed at the same level. Unlike RNA-Seq, Ribo-Seq only targets mRNA sequences protected by the ribosome during the process of decoding by translation, which unveils the composition and regulation of the expressed proteome.
With the strength of detecting the transcription and translation of all genes in the cell at the same time, Ribo-Seq is able to detect cells infected with the virus, leading to the potential discovery of novel viral proteins and improving the database of viral genome annotation. Meanwhile, this technology can detect the dynamic changes of the translation level of the host after being infected by the virus, providing clues for studying the interaction between the virus and the host.
Ribo-Seq in Virology
In 2012, researchers first used Ribo-Seq in virology research. And in the following years, more virology studies based on Ribo-Seq have explored the areas of novel ORFs discovery, translation mechanism analysis, dynamical analysis of virus replication processes, and detecting host gene expression regulation, bringing new knowledge on different viruses.
ORFs discovery: Ribo-Seq improved genome annotation, initially carried out in DNA viruses with relatively large and complex genomes. Improved genome annotation made obtaining the expressing virus DNA from the host cell laterally possible. Many previously undiscovered ORFs had then been found. And novel ORFs have also been discovered in viruses that are considered well-studied. Ribo-Seq has greatly enriched the database of viral genomes and improved the quantity and quality of the viral genomes, which are of great significance for the study of virology.
Translation mechanism analysis: More information of the translation mechanism has been revealed by Ribo-seq. For instance, among most of the new ORFs found in HCMV (human cytomegalovirus), CUG serves as the start codon, which suggests that these non-typical start codons may be important in viral gene expression. In addition, a large number of ORFs on lncRNAs are very short and efficiently translated, but no corresponding peptide/protein products can be found, suggesting that the translation of these ORFs is a means of regulation.
Dynamical analysis of virus replication processes: The analysis of viral genome replication and protein expression at different time points is a scientific concern to many researchers, and is particularly necessary for viruses with varied gene expression strictly according to different stages of the life cycle.
Detecting host gene expression regulation: Viruses are completely dependent on the host cellular ribosome for translation. Viruses have evolved ways to take advantage of the cells they infect and use them as translation machines, which might lead to a process called Host Shutoff – the inhibition of host gene expression, a common process during viral infection. Ribo-Seq can be performed at both mRNA and translation levels, giving an overall picture of the cell and infection.
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