Getting an accurate picture of the real-time transcriptional activity of a cell: This is the goal of a new research project at the University of Würzburg which is funded by the European Research Council.
If you paid attention during biology lessons, you may remember that genetic information in human cells is contained in the cell nucleus as a DNA double helix. When a gene is activated, this information needs to get out of the nucleus to allow the cell to take further action. This is where RNA comes into play: It transfers the genetic information from the DNA to the sites of protein biosynthesis in living cells.
Economical use is the goal
So in order to know which genes are active inside a cell at a given time, you have to determine with pinpoint accuracy which RNA molecules are being actively synthesized at any given moment. This allows conclusions to be drawn as to the corresponding genes and proteins. The required technique already exists; it is called high-throughput RNA sequencing. Its development has revolutionised basic biomedical research in the past decade. It enables pinpointing the activity of thousands of genes simultaneously at RNA level to better recognise and understand the changes taking place during diseases, for example.
Researchers of the University of Würzburg are now working to improve this analysis technique to allow time-resolved analysis of RNA synthesis. Their goal is to develop and provide a new analysis platform needed for this purpose. Professor Lars Dölken, who holds the Chair of Virology, and Florian Erhard, a junior professor of systems virology at the same department, both participate in the project. The European Research Council now approved an ERC Proof of Concept Grant worth EUR 150,000 to fund their research.
Telltale block exchange
"T-GRAND-SLAM: Translating GRAND-SLAM for hHigh temporal resolution measurements of cellular transcriptional activities" is the exact title of the research project conducted by the two virologists. For their work, they use three approaches developed in the past two years, which have substantially enhanced the temporal resolution performance of RNA sequencing techniques. Dölken explains the underlying principle as follows: "Cells in culture are provided with a modified RNA building block for a short time, the so-called 4-thiouridine (4sU) instead of normal uridine." The cells absorb the 4sU efficiently and integrate it in all newly transcribed RNA molecules at a rate of about 1 in 50.
Subsequently, the integrated 4sU molecules can be biochemically converted to another RNA block, a cytosine. The scientists then use high-throughput sequencing on the corresponding samples to determine alterations in the overall RNA profile of the corresponding cells for more than 10,000 genes. Moreover, the observed exchange processes of 4sU molecules to cytosine enable them to measure the percentage of "new" RNA molecules based on the uridine-to-cytosine exchanges.
Vast amounts of data call for new methods
New bioinformatics procedures are needed to analyse the huge amounts of data. This is where Dölken and Erhard recently accomplished a major breakthrough. Instead of simply counting the uridine-to-cytosine exchange processes for each gene in the collected data as done previously, they developed a computational approach (GRAND-SLAM: Globally Refined Analysis of Newly transcribed RNA and Decay rates using SLAM-seq) which accounts for the 4sU integration rates, sequencing errors and special stochastic effects in order to determine the percentage of new to old RNA molecules for each gene with high precision.
In 2018, the two researchers applied for a patent for their approach. For example, GRAND-SLAM allows them to identify if the contribution of newly synthesized RNA molecules of any given gene spikes from 25 to 75 percent in the first two hours of a herpes virus infection. Such short-term changes were previously not yet visible in total cellular RNA. They now aim to translate GRAND-SLAM into an analysis pipeline to be used by reseachers and industry worldwide.
Preliminary work in a second ERC project
The associated efforts are ongoing in a second research project for which Dölken received another grant from the European Research Council in 2016, a EUR 2 million ERC Consolidator Grant. Within the scope of this project, Dölken and his team study the molecular mechanisms used by herpes simplex virus type 1 to reprogram human cells to its advantage. Their main goal is to shed light on the RNA metabolic processes taking place inside human cells. To accomplish this, Florian Erhard designs new bioinformatics procedures required to optimally analyse and compare the large amounts of data.
The virologists intend to use the money from the Proof of Concept Grants to design analysis pipelines for their technique and extend them to new applications. The researchers hope that the enhanced analysis techniques will deliver fundamentally new insights in all domains: from basic biomedical research to translational projects whose results are used directly for patient treatment.
Proof of Concept Grant
A Proof of Concept Grant can only be applied for by scientists who already have an ERC Grant and want to turn their research output into a commercially valuable proposition in a pre-commercial phase.
A Proof of Concept Grant should aim to verify the market potential of such an idea. With this follow-up grant the ERC hence funds further development activities with regard to the viability, commercialisation or marketing of an idea.