mRNA regulation of actin binding proteins

Project Information

mRNArABP

Grant agreement ID: 101180603

DOI

10.3030/101180603

Project closed

EC signature date 29 April 2024

Start date 1 July 2024

End date 30 June 2026

Funded under

• Widening participation and spreading excellence

Total cost

No data

EU contribution

€ 155 559,36

Coordinated by

KEMIJSKI INSTITUT
Slovenia

Periodic Reporting for period 1 - mRNArABP (mRNA regulation of actin binding proteins)

Reporting period: 2024-07-01 to 2026-06-30

Summary of the context and overall objectives of the project

Changes in animal cell shape are essential during key cellular processes such as cell division, migration, and tissue morphogenesis. Animal cell shape is primarily maintained by the actin cytoskeleton, a dynamic network of actin filaments, myosin motors, and numerous actin-binding proteins (actin-BPs) attached to the cell membrane. Disruptions in the homeostasis of any of these components can lead to various disease states. For example, disruptions in cell division and/or migration are observed in multiple cancers, while defects in cell migration can negatively impact an organism's immune response. Maintaining specific cell shapes is also important for proper neuronal function. Understanding how the levels of actin and actin-BPs are maintained is therefore key to understanding how healthy cellular physiology is preserved.
While regulation of actin networks is often considered at the level of protein activity, cells can also control cytoskeletal organisation earlier through the regulation of messenger RNAs (mRNAs). mRNAs are not only passive templates for protein synthesis; their localisation, stability, nuclear export, and translation can be regulated in time and space, allowing cells to produce proteins where and when they are needed. This spatial regulation of mRNAs is an established mechanism for locally tuning gene expression and is particularly relevant for proteins that function within defined subcellular structures, such as the actin cytoskeleton.
Recently, a novel molecular mechanism termed interstasis was identified, in which similarities in mRNA sequences lead to their co-regulation and the coordinated regulation of the proteins they encode. In particular, when their levels increase, low-complexity domain (LCD) proteins encoded by mRNAs containing repetitive sequence motifs (multivalent mRNAs) become co-regulated through protein and mRNA retention within nuclear speckles, together with their own mRNAs and other mRNAs encoded by similarly multivalent sequences. This regulatory mechanism was particularly demonstrated for multivalent mRNAs containing repetitive GA motifs. Interestingly, many actin-BPs are encoded by mRNAs containing C-rich multivalent regions. Therefore, we asked whether the C-rich multivalency of mRNAs encoding actin-BPs enables their co-regulation during disruptions of actin networks and thereby ensures robust control of cell shape.
In this project, we investigated how actin-BPs are regulated at the RNA level. We specifically focused on proteins encoded by C-rich multivalent mRNAs and asked whether similarities in mRNA sequence lead to their co-regulation. These findings contribute to our understanding of fundamental principles of mRNA regulation and how key cellular processes are controlled. In the longer term, this project may contribute to the development of new diagnostic and therapeutic approaches for diseases associated with cytoskeletal dysfunction.

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

To better understand how genes that produce actin-binding proteins (proteins involved in maintaining cell structure and movement) are regulated, we combined computational analyses with laboratory-based experiments.
We first used bioinformatics approaches to identify common sequence patterns in the messenger RNAs (mRNAs) that encode actin-binding proteins, focusing on those containing regions rich in the nucleotide cytosine (C). We then analysed these sequences and the proteins they produce to assess their potential to form molecular assemblies or condensates within cells.
Next, we carried out a series of experiments to test how these mRNAs respond to different cellular conditions. These included increasing the levels of selected C-rich RNA sequences and disrupting the normal dynamics of the actin cytoskeleton. We examined whether these changes affected the ability of candidate mRNAs to form condensates, which could indicate a previously unrecognised regulatory mechanism.
Finally, we investigated the role of specific RNA-binding proteins that interact with C-rich RNA sequences. By reducing the levels of these proteins in cells, we assessed their impact on cell division and cellular function.
Overall, this work combined computational biology, microscopy, and molecular biology techniques to provide new insights into how mRNAs encoding actin-binding proteins may be regulated within cells. The results contribute to a better understanding of the molecular mechanisms that control cell organisation and behaviour.

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)

In this project, we examined the regulation of actin-binding proteins at the mRNA level. In the first part of the study, we performed bioinformatic analyses to determine whether these mRNAs are multivalent and to identify the predominant forms of multivalency. We found that mRNAs encoding actin-BPs are predominantly CG-, AG-, or C-enriched. We focused primarily on C-enriched mRNAs.
Interestingly, many proteins encoded by these mRNAs are predicted to be condensation-prone, and most are predicted to act as drivers of biomolecular condensation. Furthermore, analysis of their representation in different cellular condensates revealed enrichment within presynaptic clusters and postsynaptic densities. These findings suggest a promising avenue for future applications of this work in neuroscience and neurodegeneration research.
Overall, we identified more than 128 actin-BPs encoded by C-rich mRNAs. Among these, we selected several candidates known to play important roles in actin polymerisation, including members of the WAS protein family. Experimentally, we first focused on identifying conditions that alter the behaviour of mRNAs encoding these candidates. Specifically, we examined changes in localisation and condensation using light microscopy. We investigated how stabilisation or inhibition of actin polymerisation, as well as overexpression of C-rich sequences, affected these mRNAs. Interestingly, most experimental conditions did not significantly alter the localisation or condensation of these candidate mRNAs or other transcripts examined.
Overall, the experimental data suggest that C-rich multivalency may not drive mRNA condensation directly but instead may contribute to regulation through an alternative mechanism.