Mechanisms of human mRNA packaging and export

Project Information

mRNApackex

Grant agreement ID: 101150910

DOI

10.3030/101150910

Project closed

EC signature date 20 March 2024

Start date 1 May 2024

End date 30 June 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 199 440,96

Coordinated by

FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
Austria

Periodic Reporting for period 1 - mRNApackex (Mechanisms of human mRNA packaging and export)

Reporting period: 2024-05-01 to 2026-06-30

Summary of the context and overall objectives of the project

Messenger RNAs carry genetic information from DNA to the cellular machinery that produces proteins. Before an RNA can be used, it must be correctly processed and exported from the nucleus. Defective or incomplete RNAs should instead be retained and degraded. Cells therefore need a reliable way to distinguish between RNAs that are ready for export and those that are not.

mRNApackex investigated whether this decision is controlled by the way an RNA is packaged with proteins into a messenger ribonucleoprotein particle, or mRNP. The project had three main scientific objectives: to determine how mRNA packaging affects nuclear export and degradation; to use a genome-wide screen to identify proteins capable of promoting export; and to characterise promising export factors in more detail.

The project combined time-resolved RNA sequencing, mathematical modelling, rapid protein depletion, metabolomics and super-resolution RNA imaging. Its main pathway to impact is through improved methods, reusable software and quantitative datasets that can help researchers study RNA production, processing and turnover more accurately. In the longer term, a better understanding of nuclear RNA quality control may support research into viral RNA, gene-expression defects in cancer and other diseases involving RNA-processing pathways. The project was focused on fundamental molecular biology and did not require the integration of social sciences or humanities.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The main part of the project used time-resolved RNA sequencing to measure how quickly newly produced RNAs are exported from the nucleus or degraded there. Early experiments revealed that commonly used RNA-labelling conditions introduced a technical bias during sequencing. Further experiments identified a second bias caused by the gradual accumulation of the labelled molecule inside cells.

To address this problem, the project developed RoSA-MoSA, a computational method that estimates this accumulation directly from sequencing data and includes it in the analysis. The computational estimate closely matched an independent measurement made using metabolomics. This provides confidence that the method is correcting a real biological effect rather than simply improving the fit of the model.

Using optimised conditions, approximately 5,000 genes were modelled. The approach was also applied to a dataset covering approximately 50,000 introns, allowing different stages of RNA splicing to be measured. These analyses showed that completing splicing accounts for a substantial part of the time an RNA spends in the nucleus and can be the rate-limiting step for export.

The project also examined the effects of depleting EIF4A3, a protein deposited on RNA during splicing, and NXF1, a central RNA-export factor. Depleting EIF4A3 reduced the average export rate of intron-containing RNAs by approximately 50% and also reduced their nuclear stability. Super-resolution RNA imaging was used as an independent way to investigate these findings.

The planned genome-wide ORF-tag screen could not be completed. The reporter worked in human cells, whereas the available screening system worked in mouse embryonic stem cells, where the reporter did not behave as required. Attempts to transfer the screening system to human cells did not achieve sufficient efficiency. The remaining effort was therefore redirected towards the productive sequencing, modelling, protein-depletion and imaging work. Manuscripts are now in preparation, and the associated software, analysis code and sequencing data are being prepared for public release.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

RoSA-MoSA addresses a previously unresolved source of systematic error in SLAM-seq, a widely used method for measuring RNA production and turnover. Without accounting for the gradual accumulation of the labelled molecule inside cells, early labelling is underestimated and the calculated turnover of short-lived RNAs can be biased. The accumulation rate estimated by RoSA-MoSA was 0.0129 per minute, compared with 0.0131 per minute measured independently by metabolomics. This close agreement provides quantitative validation of the method.

The biological results also refine the existing model of how splicing and RNA packaging affect nuclear export. Incomplete splicing delays export, but once splicing is complete, RNAs with more introns—and therefore more exon junction complexes—are exported more quickly. The EIF4A3 depletion experiments further indicate that this form of RNA packaging supports both efficient export and protection from nuclear degradation.

These findings provide a more quantitative view of how RNA processing and packaging influence RNA fate. Potential users include researchers studying RNA kinetics, splicing, nuclear export, metabolic RNA labelling and mRNP structure.

Further uptake will require publication and independent testing of the method in additional cell types and labelling conditions. RoSA-MoSA and the supporting analysis workflows will be released through GitHub with documentation and reproducible examples. Raw and processed sequencing data, metadata and derived kinetic parameters will be deposited in GEO. The repositories will be linked to the corresponding preprints and publications so that other researchers can reproduce, test and extend the work.