Transcriptional Reporter Tracks Non-Coding RNA

Researchers in Germany have developed a new molecular biology tool that overcomes challenges to tracking the generation of non-coding RNA and offers advantages for studying coding RNA. Called INSPECT (Intron-encoded Scarless Programmable Extranuclear Cistronic Transcript) and developed at Helmholtz Munich and Technical University of Munich (TUM), the minimally invasive reporter system could greatly advance understanding of RNA regulation and, in turn, development of molecular therapies.

For cellular processes, genetic information is transcribed into RNA, which then undergoes further processing before it either serves as a blueprint for proteins or performs a cellular function itself. The types and quantities of RNA produced reveal a lot about the condition of cells. 

When DNA molecules are translated into proteins via RNA, researchers can track the process with existing reporter systems. However, not all human genes encode proteins. The majority of human genes is non-coding, including genes for long non-coding RNAs (lncRNA)—RNA molecules with more than 200 building blocks that do not act as blueprints for proteins. Instead, they control important processes in cells. Initial research shows that lncRNA is involved in such processes as regulating RNA production, the organization of structures in the cell nucleus or in switching certain enzymes on and off.

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

The INSPECT system, developed by a team led by Gil Westmeyer, Professor of Neurobiological Engineering at TUM and the Director of the Institute for Synthetic Biomedicine at Helmholtz Munich, offers a solution. Unlike previous methods, INSPECT encodes sequences for reporter proteins in modified introns.

“These are sequences in the pre-mature RNA molecule that are removed naturally and eliminated by the cell during processing,” says first author Dong-Jiunn Jeffery Truong. “INSPECT stabilizes the introns such that, rather than being degraded after removal, they are transported to the cellular cytoplasm where they are translated into reporter proteins.”

The researchers can then use conventional methods to detect reporter protein signals such as fluorescence.

The new molecular biology tool thus not only solves the problem of tracking the generation of non-coding RNA, but also offers advantages for studying coding RNA. Current reporter systems often run the risk of damaging the RNA or proteins under investigation because they must be fused directly to the RNA being studied in order to be co-translated into proteins. Rather than modifying the completed RNA or the proteins, INSPECT modifies the 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 larger quantities in response to infections. They have 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 turning into cancer cells,” says Prof. Westmeyer. “In combination with the minimally invasive reporter system EXSISERS, which we previously developed to study protein isoforms, it may be possible in the future to study an entire genetic regulation process from RNA processing to the production of specific protein variants in living cells.”

INSPECT is described in more detail in Nature Cell Biology.