New Sequencing Technique Overcomes Limitations of Ribo-seq

A research team at the University of Würzburg (JMU) has developed a technique to efficiently gain detailed information about the genetic activity of individual cells. Name INRI-seq—short for in vitro Ribo-seq—the study authors believe the breakthrough will help in the development of new, targeted therapies.

"We have developed a technique that can be used to analyze the translational landscape of a fully customizable synthetic transcriptome, in other words one outside the cell," says Jörg Vogel, head of the Institute for Molecular Infection Biology at JMU and Director of Helmholtz Institute for RNA-based Infection Research (HIRI). 

A transcriptome is a collection of all the genes that are active in a cell at a given point in time. It consists of the sum of the existing mRNA, which transport the blueprints for proteins from the cell nucleus to the ribosomes. Ribosomes are the "protein factories" of the cell, where translation of the nucleotide sequence of the mRNA into the amino acid sequence of a protein takes place.

In principle, INRI-seq is a refinement of comparable methods that pursue the same goal but provide less accurate results or have other disadvantages. For example, RNA sequencing (RNA-seq) determines the concentration of mRNA in cells, allowing conclusions to be drawn about their active genes. However, the final protein abundance does not always correlate with the respective mRNA concentrations.

A more accurate technique is ribosome profiling (Ribo-seq). Over the past ten years, this has become one of the main methods for measuring protein synthesis directly in a transcriptome-wide manner. "While Ribo-seq has greatly advanced the study of translation-related processes, the method has not been without limitations," says Vogel.

For example, detecting weakly expressed genes with Ribo-seq is a major challenge, and in common study designs many genes end up not being recorded. Similarly, a Ribo-seq study of microbes from important ecological habitats such as the human gut is difficult since many of them cannot be cultured in the laboratory.

Another shortcoming, according to Vogel, is the fact that "on the mechanistic level, Ribo-seq-based studies of molecules affecting translation, such as special antibiotics, can be hampered by cellular responses." Since Ribo-seq is performed on living cells, it can be difficult to distinguish between direct and indirect effects on translation.

To overcome some of these limitations, the scientists from Würzburg have developed INRI-seq for the global study of translation in a cell-free environment. INRI-seq uses a commercially available in vitro translation system combined with an in vitro-synthesized, fully customizable transcriptome that allows better control of individual mRNA levels. "With INRI-seq, for example, it is no longer necessary for translation-modulating substances to traverse cellular membranes or to extract ribosomes from a large number of living cells," says Vogel, outlining the advantages of the technique. "You also need a lot less of the often expensive substance that you want to study, such as a new antibiotic that can only be produced on a small scale. INRI-seq therefore also saves time and money."

To demonstrate their technique's efficacy, the team used a synthetically generated transcriptome of the bacterium Escherichia coli. Compared to a technically similar study on living cells, INRI-seq identified almost four times more sites where translation processes are initiated, demonstrating its high sensitivity.

 "INRI-seq bears great potential as an alternative method for studying translation process and thus also substances that can influence these processes," says Vogel.

The technique is described in more detail in the journal Nucleic Acids Research.