DEAD-box ATPases as master regulators of phase-separated compartments to control cellular RNA flux and the remodeling of RNA-protein complexes

Project Information

DDX TRANSIT

Grant agreement ID: 950262

DOI

10.3030/950262

EC signature date 9 September 2020

Start date 1 February 2021

End date 31 January 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 845,00

EU contribution

€ 1 499 845,00

Coordinated by

UNIVERSITAT BASEL
Switzerland

Periodic Reporting for period 3 - DDX TRANSIT (DEAD-box ATPases as master regulators of phase-separated compartments to control cellular RNA flux and the remodeling of RNA-protein complexes)

Reporting period: 2024-02-01 to 2025-07-31

Summary of the context and overall objectives of the project

Life fundamentally relies on the precise control of gene expression, necessitating the orderly and efficient processing of diverse RNA molecules. Messenger RNAs (mRNAs), enveloped in a dynamically evolving coat of passenger proteins, transit from their transcription in the nucleus to translation in the cytoplasm and ultimately decay. The importance of mRNA research came to the forefront with the rapid emergence of mRNA-based vaccines in response to the COVID pandemic. Similarly, ribosomal rRNAs migrate through the nucleolus where they gradually encounter ribosomal proteins to assemble functional ribosomes in the cytoplasm. However, our understanding of the intricate processes governing this spatiotemporally orchestrated RNA flux are still limited.

Intriguingly, numerous RNA processing events are associated with membraneless organelles formed by biomolecular condensation, such as stress granules (for mRNA) or the nucleolus (for rRNA). However, the functional role of condensates in RNA processing remains elusive. We have discovered that the family of DEAD-box ATPases (DDXs) operates as master regulators of RNA-containing membraneless organelles, spanning the evolutionary spectrum from bacteria to humans. We postulate that DDXs employ their condensation and ATPase activities to govern condensate dynamics, and speculate that they thereby influence the flow and processing of RNA molecules in the cell.

In this project, we have three main objectives. First, we want to characterize the role of candidate DDXs in governing the flux of mRNA molecules, and utilize specific DDX mutants as unique tool to halt RNA flux and elucidate changes in passenger protein composition. Furthermore, we aim to dissect how DDXs facilitate the efficient flow of rRNA during ribosome assembly and concomitantly influence dynamics of the nucleolar condensate environment. And third, we want to unravel the functional role of DDX condensates as biomolecular filters, selectively enriching or excluding proteins, and how this selectivity contributes to the dynamic remodeling of the RNA-protein coat and the directional flow of RNA.

Our research will provide key novel insight into our understanding of RNA processing and uncover novel layers of gene expression regulation.

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

For WP 1, we have achieved the design and identification of mutants capable of halting mRNA flux. We are currently in the process of establishing a protocol to detect alterations in passenger protein composition and sequencing enriched mRNAs while actively exploring underlying regulatory mechanisms.

For WP 2, we have successfully discovered nucleolar DDXs that can form condensates and started characterizing the biophysical properties of these condensates. We have also generated separation-of-function mutants for ATPase and condensation activities and are currently constructing yeast strains to assess the impact of these mutants on rRNA maturation and nucleolar structure.

In WP 3, we have established an innovative method to quantify protein enrichment within condensates. This approach has been applied to nucleolar DDXs thus far, with plans to extend to mRNA-associated condensates in the near future.

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)

This project operates at the intersection of RNA processing and biomolecular condensates, marking a departure from the current state of the art in the field in multiple dimensions. First, it emphasizes a rigorous investigation into the functional aspects of condensates, adopting a mechanistic perspective. By integrating biochemistry and cell biology, this project aims to mitigate potential artifacts in its findings. And furthermore, a recent shift in focus towards regulation, rather than solely assembly, furthermore addresses the functional role of condensation for RNA processing.

By project completion, we anticipate a significant advancement in our comprehension of DEAD-box ATPases (DDXs) as pivotal regulators of RNA-containing membraneless organelles in mRNA and rRNA processing. We also aim to unveil the mechanisms through which DDX condensates impact the protein composition and dynamic remodeling of the RNA-protein coat, ultimately enhancing our understanding of RNA processing and gene expression regulation.