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ORFtag: innovative method tags and tracks proteins genome-wide

05 Jul 2024
Filip Nemcko.

Filip Nemcko, Research Associate in the lab of Alexander Stark, completed his PhD in April 2024. During his doctoral studies in the Vienna BioCenter PhD Program, he co-developed ORFtag–a new method that makes it easier to study the function of proteins by mass tagging and tracking them across the entire genome. The study, a collaborative effort of Vienna BioCenter scientists from the IMP, the Institute of Molecular Biotechnology (IMBA), and the Max Perutz Labs, is now published in the journal Nature Methods. In this interview, Nemcko reflects on why this method is a breakthrough–and how it will now be applied.

Could you briefly outline what the main idea behind ORFtag is?

ORFtag gets its name from “Open Reading Frames”, or ORFs, specific sections of DNA that have the potential to produce proteins. Nearly every process in our bodies relies on proteins, so understanding what each one does is important for understanding life. The main idea behind ORFtag was to develop a method that allows to screen and functionally characterise all mammalian proteins. Our genome contains instructions for around 20,000 proteins, but the roles of many are still not well understood, with about 20 percent having completely unknown functions. In our work, we developed and used ORFtag to identify proteins that control the production of other proteins on different levels, specifically that act as transcriptional activators, repressors, and post-transcriptional regulators. However, ORFtag can be adapted to investigate any biological question related to protein functions.

How does it work?

With this method a 'tag' is attached to each protein by leveraging a retrovirus—a type of virus that can sneak its genetic material into the DNA of the cells it infects. This allows us to insert a small DNA segment into the cell's genome, which produces a 'tag' attaching to newly formed proteins, making them easier to find and study. These tags not only help us track the proteins, but also allow us to manipulate their behaviour, such as directing them to specific parts of the cell. By testing these tagged proteins, we can see if they perform certain functions, for example turn genes on or off.

What’s the main difference to existing approaches?

Conceptually, there are methods that test for loss-of-function and involve deactivating genes to study their effects. These are CRISPR and RNAi screens for example, which knock out or silence genes. However, they cannot directly test the proteins’ function–for example they can identify that the proteins are important for gene expression, but cannot reveal whether they work at transcription, translation, or other levels. An alternative to these methods are sufficiency-based screens, which directly observe protein function. These methods are typically very laborious, and this is where ORFtag steps in–it’s a quick and easy protocol.

What specific advantages does it offer compared to other methods?

It is faster, cheaper, and simpler to use in the lab. Unlike traditional approaches such as assembling the ORFeome collection–a complete set of ORFs in a genome–which can take decades, and be expensive to purchase and manage, ORFtag simply involves infecting cells with a virus, making it highly efficient and cost-effective. Moreover, this method can be applied across various model organisms. Researchers using ORFtag can conduct screens in just a matter of days, achieving results much more quickly compared to conventional methods.

What can researchers do with ORFtag next?

There are countless possibilities. In general, with this method researchers can target and explore functions of specific proteins of interest. This enables scientists to investigate and understand how proteins work in various biological processes, opening the way for several applications in diverse areas of research.

How did the collaboration between the institute's contribute to the development of this method?

I think this is the kind of project that could have only happened at the Vienna BioCenter. The broad collaboration between the institutes was crucial to developing ORFtag. Each research group brought different expertise to the table: the Ameres lab focused on RNA metabolism, our lab with Alexander Stark on transcriptional activation, and Brennecke's on transcriptional repression and silencing. This complementary approach allowed us to integrate diverse perspectives, leading to the successful development and use of ORFtag.

And what was the most rewarding aspect of taking part in this research project?

For me, the most rewarding aspect of participating in this research project has been seeing the interesting results from our screens. It was exciting to see our method successfully identifying both known and unknown proteins–while the known proteins validated our approach, the interesting proteins are ones that were previously never linked to the respective processes. Currently, I'm characterising these proteins further, which is a very exciting phase of our research.

Original publication

Filip Nemčko*, Moritz Himmelsbach*, Vincent Loubiere, Ramesh Yelagandula, Michaela Pagani, Nina Fasching, Julius Brennecke, Ulrich Elling, Alexander Stark, Stefan L. Ameres “Proteome-scale tagging and functional screening in mammalian cells by ORFtag”.
Nature Methods, DOI: 10.1038/s41592-024-02339-x.
*These authors contributed equally.

Further reading

Lab of Alexander Stark

Lab of Stefan L. Ameres

Lab of Julius Brennecke

Lab of Ulrich Elling

Full news item on Vienna Biocenter website

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