Biological tool for tracking cellular processes

For cellular processes, genetic information in the form of DNA is first transcribed into RNA. This is then processed before it either serves as a building instruction for proteins or takes on a function in the cell itself. How much and which RNA is currently being produced says a lot about the state of a cell. For example, during inflammation, cells produce more RNA that codes for the proteins of the immune response.

When genes are further converted into proteins via RNA, researchers can track this with the help of known reporter systems. However, only some of the human genes code for proteins. A large proportion of human genes are non-coding, including genes for so-called long non-coding RNAs (lncRNA). These are RNAs with more than 200 building blocks that do not serve as building instructions for proteins, but instead control important processes in the cell as molecules themselves. According to initial findings, they are involved, for example, in the regulation of RNA production, the organisation of structures in the cell nucleus or in switching certain enzymes on and off.

Despite their importance for cellular processes, lncRNAs could only be investigated with difficulty and with limitations using previous methods, for example for fixed cells at individual time points, as classic reporter systems based on the conversion to proteins cannot be used for this purpose.

Monitoring of non-coding RNA

A team led by Gil Westmeyer, Professor of Neurobiological Engineering at TUM and Director of the Institute of Synthetic Biomedicine at Helmholtz Munich, presents a newly developed reporter system, called „INSPECT“, in the journal „Nature Cell Biology“.

„Unlike previous methods, Inspect encodes the sequences for reporter proteins in modified introns. Introns are sections of unfinished RNA that are naturally separated and degraded by the cell during processing. However, Inspect stabilises the introns in such a way that they are not degraded after separation, but are brought into the cell cytoplasm and the sequences are converted into reporter proteins“, explains first author Dong-Jiunn Jeffery Truong. The researchers can then detect the signal of the reporter proteins, for example fluorescence, as with conventional methods.

Coding RNA

The molecular biology tool developed not only solves the problem of how to track the production of non-coding RNA, but also brings advantages for the investigation of coding RNA. Previous reporter systems often harbour the risk of damaging the RNA or proteins being searched for, as they have to be placed directly adjacent to the RNA to be examined, for example, in order to be converted into proteins in the process. In contrast, the Inspect system only modifies the introns, but not the finished RNA or the proteins.

The team has already been able to confirm the functionality of Inspect using various examples of coding and non-coding RNA. For example, they used it to track the production of RNA for interleukin 2, a protein that is increasingly produced during inflammation. They were also able to monitor the production of two lncRNAs with high sensitivity and track changes in regulation during the period under investigation.

„Inspect adds an important molecular biology tool to the biomedical toolbox. It can be used to easily investigate how certain non-coding RNA molecules are involved in cell development and how their regulation can possibly be changed, for example to prevent transformation into cancer cells," says Prof Dr Westmeyer. „Together with our previously developed reporter system „EXSISERS“, which uses a similarly minimally invasive principle for proteins, an entire gene regulation process from RNA processing to the production of specific protein forms in living cells could be investigated in the future.“

Source: LABO from 14 November 2022