Molecular monitoring of RNA regulation

Molecular monitoring of RNA regulation

−1dox Bars represent the mean of three biological replicates, with error bars representing SD. Statistical significance was determined by one-way ANOVA with Bonferroni’s multiple comparison test (*P<0.05; ***P<0.001; ****P<0.0001) . Recognition: nature cell biology (2022). DOI: 10.1038/s41556-022-00998-6″ width=”800″ height=”502″/>
INSPECT reporter enables the modular readout of coding genes with RNA reporters. a, To demonstrate the alternative use of INSPECT transcripts as secreted RNA barcode reporters, a Dox-inducible construct expressing msfGFP along with an INSPECT reporter module was generated. b, Instead of the translation unit with an IRES reporter CDS, INSPECT encodes an RNA reporter followed by a 5× PP7 aptamer motif. As a reference, mScarlet-I was constitutively expressed from a second plasmid along with a second RNA reporter. A synthetic chimera of HIV-1 gag polyprotein and PCP was used to secrete PP7-tagged RNA into the extracellular environment via virus-like particle (VLP) budding. c, A viability/cytotoxicity assay was performed 48 h after transient transfection with an expression plasmid encoding mouse Pgk1 promoter-driven HIV-1 gag. Bars represent the mean of n = 3 biological replicates, with error bars sd, d, e, quantification of fluorescence signals from msfGFP (d) and mScarlet-I (e) and the corresponding INSPECT RNA reporters 48 h after transfection and Show 24 h after induction with the indicated Dox concentrations. The mean fluorescence intensity was corrected for background fluorescence. INSPECT RNA reporter levels were normalized to maximal induction at 1 ng ml−1dox Bars represent the mean of three biological replicates, with error bars representing SD. Statistical significance was determined by one-way ANOVA with Bonferroni’s multiple comparison test (*P<0.05; ***P<0.001; ****P<0.0001) . Recognition: nature cell biology (2022). DOI: 10.1038/s41556-022-00998-6

The better we understand cellular processes such as RNA regulation, the better molecular therapies can be developed. So far, it has been particularly difficult to track the regulation of non-coding RNA, i.e. the RNA that is not further converted into proteins.

A research team from the Technical University of Munich (TUM) and Helmholtz Munich has now developed a minimally invasive reporter system that enables highly sensitive monitoring of RNA production from both coding and non-coding RNA.

For cellular processes, our DNA genetic information is transcribed into RNA, which is then processed further before serving as either the blueprint for proteins or performing a cellular function itself. Which types of RNA are produced and in what amounts reveals a lot about the state of our cells. For example, when infected, 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 follow the process using existing reporter systems. However, not all human genes encode proteins. The majority of human genes are non-coding, including long non-coding RNA (lncRNA) genes. These are RNA molecules with more than 200 building blocks that do not act as a blueprint for proteins. Instead, they control important processes in cells. Initial investigations show that lncRNA is involved, among other things, in the regulation of RNA production, the organization of structures in the cell nucleus or the switching on and off of certain enzymes.

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

INSPECT allows monitoring of non-coding RNA

A solution has now been found in the form of a new reporting system: INSPECT. A team led by Gil Westmeyer, Professor of Neurobiological Engineering at TUM and Director of the Institute for Synthetic Biomedicine at Helmholtz Munich, has now published the newly developed reporter system in the journal nature cell biology.

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

INSPECT does not change either the finished RNA or the proteins

The new molecular biology tool not only solves the problem of tracing the formation of non-coding RNA, but also offers advantages for the investigation of coding RNA. Current reporter systems often run the risk of damaging the RNA or proteins under study because, for example, they have to be fused directly to the RNA under study in order to be co-translated into proteins. Instead of modifying the finished RNA or the proteins, INSPECT modifies the introns.

The team demonstrated the function of INSPECT using different examples of coding and non-coding RNA. They tracked the production of RNA for interleukin 2, a protein that is produced in larger amounts in response to infection. They also achieved highly sensitive monitoring of the production of two lncRNAs and tracked changes in regulation over the study period.

“INSPECT adds an important molecular biological tool to the biomedical toolbox. It makes it easier to study the role of certain non-coding RNA molecules in cell development and how their regulation can be modulated, for example to prevent them from developing in cancer cells,” says Prof. Westmeyer.

“In combination with the minimally invasive reporter system EXSISERS, which we previously developed to study protein isoforms, it could in future be possible to study an entire genetic regulatory process from RNA processing to the production of specific protein variants in living cells.”

More information:
Dong-Jiunn Jeffery Truong et al, Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and noncoding RNAs, nature cell biology (2022). DOI: 10.1038/s41556-022-00998-6

Dong-Jiunn Jeffery Truong et al, Non-invasive and high-throughput query of exon-specific isoform expression, nature cell biology (2021). DOI: 10.1038/s41556-021-00678-x

Provided by the Technical University of Munich

Quote: Molecular Monitoring of RNA Regulation (2022, November 14), retrieved November 14, 2022 from

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