Minimally invasive reporter system allows highly sensitive monitoring of RNA production

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The better we understand cellular processes such as RNA regulation, the better molecular therapies can be developed. Until now, it has been especially difficult to trace the regulation of non-coding RNA, which is RNA that is not further converted into protein. A research team from Helmholtz Munich and the Technical University of Munich (TUM) have now developed a minimally invasive reporter system that enables highly sensitive monitoring of both coding and noncoding RNA production.

For cellular processes, our genetic information from DNA is transcribed into RNA, which then undergoes further processing before it serves as a blueprint for proteins or performs a cellular function of its own. What types of RNA are produced and in what amounts reveals a lot about the state of our cells. In the event of an infection, for example, cells produce increased amounts of RNA molecules that code for proteins involved in the immune response.

When DNA molecules are translated into proteins via RNA, researchers can track the process with existing reporter systems. However, not all human genes code for proteins. Most human genes do not code, including genes for long non-coding RNAs (lncRNA). These are RNA molecules with over 200 building blocks that do not act as blueprints for proteins. Instead, they control important processes in cells. Initial research shows that lncRNA participates in processes such as the regulation of RNA production, the organization of structures in the cell nucleus, or the activation and deactivation of certain enzymes.

Despite their importance to cellular processes, it has been difficult to investigate lncRNAs with existing methods. Until now, this was only partially possible, for example, in cells fixed at specific time points, because classical reporter systems based on protein translation cannot be used.

INSPECT enables the monitoring of non-coding RNA

Now a solution has been found in the form of a new reporting system: INSPECT. A team working with Gil Westmeyer, professor of neurobiological engineering at TUM and director of the Institute for Synthetic Biomedicine at Helmholtz Munich, has published the newly developed reporting system in the journal Nature Cell Biology.

“Unlike previous methods, INSPECT encodes sequences for reporter proteins in modified introns. These are sequences in the premature RNA molecule that are naturally deleted and removed by the cell during processing. INSPECT stabilizes the introns in such a way that , instead of being degraded after deletion, they are transported to the cell cytoplasm where they are translated into reporter proteins,” explains first author Dong-Jiunn Jeffery Truong. The researchers can then use conventional methods to detect signals from reporter proteins, such as fluorescence.

INSPECT does not modify full length RNA or proteins

Thus, the new molecular biology tool not only solves the problem of tracking non-coding RNA generation, but also offers advantages for studying coding RNA. Current reporter systems are often at risk of damaging the RNA or proteins under investigation, for example, because they must be directly fused with the RNA under study in order to be co-translated into proteins. Instead of modifying whole RNA or proteins, INSPECT modifies introns.

The team has demonstrated the function of INSPECT using various examples of coding and non-coding RNA. They tracked the production of RNA for interleukin 2, a protein that is produced in large amounts in response to infections. They also achieved highly sensitive monitoring of the production of two lncRNAs and tracked changes in regulation during the investigation period.

“INSPECT adds an important molecular biology tool to the biomedical toolbox. It makes it easier to study the role of certain non-coding RNA molecules in cell development and to explore how their regulation can be modulated, for example, to prevent them from become cancer cells,” says Prof. Westmeyer. “In combination with the EXSISERS minimally invasive reporter system, which we previously developed to study protein isoforms, it is possible that in the future a complete genetic regulation process, from RNA processing to the production of specific protein variants, could be studied in living cells”.

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Helmholtz Munich (Helmholtz Zentrum München Deutsches Forschungszentrum für

Magazine reference:

Dong-Jiunn Jeffery Truong, Niklas Armbrust, Julian Geilenkeuser, Eva-Maria Lederer, Tobias Heinrich Santl, Maren Beyer, Sebastian Ittermann, Emily Steinmaßl, Mariya Dyka, Gerald Raffl, Teeradon Phlairaharn, Tobias Greisle, Milica Živanić, Markus Grosch, Micha Drukker , Gil Gregor Westmeyer: Intron-encoded cistronic transcripts for minimally invasive control of sense and non-sense RNAs, Nature Cell Biology (2022), DOI: https://doi.org/10.1038/s41556-022-00998-6

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