New technology: In vitro Ribo-seq (INRI-seq) for global translational studies in a cell-free manner

Protein synthesis is one of the most energy-intensive processes in living cells, and its precise regulation becomes a key issue in
the cellular economy.
While mRNA levels determined by RNA sequencing (RNA-seq) are often used as an alternative to analyzing protein synthesis in global gene expression analysis, the final protein abundance is not always correlated with
mRNA levels.
This is due to the fact that there are also various regulatory mechanisms
such as post-transcriptional regulation of mRNA (small molecule RNA (sRNA)), translation regulation, and post-translational modification.

Over the past decade, ribosome analysis (Ribo-seq) has become the primary method
for assessing protein synthesis rates and studying translation control mechanisms at the transcriptome scope.
Ribo-seq is based on RNA-seq analysis of ribosome-protected fragments (RPFs), ribosome-binding sites on mRNA, which are preserved during
nuclease processing after cell lysis 。 Since each ribosome protective fragment represents the location of a ribosome, their sum reveals not only which mRNAs but also which parts of the coding sequence (CDS) of these mRNAs are translated, which can be used to map active translation ribosomes in vivo to provide global information about translation pauses, stalls, and translation start site use, as well as estimates
of protein copy numbers.

While Ribo-seq has greatly advanced the study of translation-related processes, the method has limitations
.
Coverage of weakly expressed genes remains challenging, making many genes—including certain gene classes with extremely low expression under normal growth conditions, such as toxins triggered only by specific pressures that cause cell death or growth inhibition—difficult to detect
.
Similarly, Ribo-seq of microorganisms from the human gut is still difficult because many of them cannot be cultured
in the laboratory.
At the mechanistic level, Ribo-seq's study of molecules that affect translation may be hampered
by cellular responses.
Furthermore, since Ribo-seq is performed on living cells, it is difficult to dissect the direct and indirect effects
on translation.

To overcome some of these limitations, a research team at the University of Würzburg (JMU) has developed a new technique: in vitro Ribo-seq (INRI-seq), for global translation studies
in a cell-free manner.
INRI-seq uses the commercially available PURExpress in vitro translation system, combined with an in vitro synthesized, fully customizable transcriptome for greater control of individual mRNA levels
.
"We have developed a technique that can be used to analyze the translation landscape of a fully customizable synthetic transcriptome, in other words, an extracellular transcriptome.


。
。 With INRI-seq, there is no longer a need to translate regulatory substances across cell membranes or extract ribosomes from large numbers of living cells," said Vogel, director of JMU's Institute for Molecular Infection Biology and lead author of the study, outlining the advantages of
the technique.
"For materials that you want to work on and are often precious, such as a new antibiotic that can only be produced on a small scale, INRI-seq requires less material, thus saving time and money
.
"

The experiment has a high success rate

The authors apply INRI-seq to the synthesis of the E.
coli transcriptome and demonstrate that the method faithfully verifies known translation start sites and can be used to predict new translation start sites
.
Compared to technically similar studies performed on living cells, INRI-seq identified almost four times more sites at the start of the translation process, showing its high sensitivity
.
Furthermore, they used this system to study the fidelity of antisense peptide nucleic acid (PNA) on translation inhibition, demonstrate mid-target specificity and define base pairing criteria
that influence the off-target effects of these short antisense oligomers.
INRI-seq has great potential as a scalable and sensitive alternative for studying translation control mechanisms and translation-regulating compounds in other organisms and biomes, including eukaryotes, to study translational initiation sites within the transcriptome without potentially confounding effects
due to ribosome extraction from living cells.
This is a significant improvement over the methods used so far, and they published their findings
in the latest issue of the journal Nucleic Acid Research.