Multivalent interactions driving RNP dynamics in development and disease

Project Information

RNPdynamics

Grant agreement ID: 835300

DOI

10.3030/835300

Project closed

EC signature date 2 December 2019

Start date 1 January 2020

End date 30 September 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 396 261,25

EU contribution

€ 2 396 261,25

Coordinated by

KEMIJSKI INSTITUT
Slovenia

Periodic Reporting for period 4 - RNPdynamics (Multivalent interactions driving RNP dynamics in development and disease)

Reporting period: 2024-07-01 to 2025-09-30

Summary of the context and overall objectives of the project

Neurodegenerative diseases like ALS and FTD afflict millions worldwide, imposing immense personal suffering, caregiving burdens, and economic costs on society. At their core, these conditions often stem from mutations in RNA-binding proteins (RBPs), predominantly within intrinsically disordered regions (IDRs) that alter protein condensation properties. While condensation has been studied through in vitro liquid droplet formation or cellular imaging of RNA granules, a critical gap persists: how these changes disrupt RNA binding functions, initiating the cascade toward neurodegeneration. Addressing this is vital, as it unlocks insights into early molecular pathology, paving the way for targeted therapies that could prevent or slow disease progression, ultimately enhancing quality of life and reducing healthcare strains.
Grasping how IDRs facilitate RNA binding is key to decoding mutation mechanisms and early disease triggers. Our research tackles this through five interconnected objectives:

Identifying roles of disorder-containing RBPs in stem cell differentiation, revealing foundational cellular processes disrupted in disease.
Evaluating multivalency in bound RNA regions for protein-RNA condensate formation, highlighting structural drivers of dysfunction.
Engineering multivalent RNA to manipulate protein-RNA assembly, offering tools for intervention.
Perturbing IDR-dependent dynamics in protein-RNA complexes to mimic and study mutational effects.
Examining species-specific multivalent complexes, providing evolutionary context for human-specific vulnerabilities.

This framework not only advances scientific understanding but holds promise for societal benefits through innovative treatments.

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 grant funded a transformative research program that deepened understanding of how intrinsically disordered regions (IDRs) in RNA-binding proteins (RBPs) regulate RNA metabolism and contribute to neurodegenerative diseases like ALS and FTD. Mutations in IDRs often alter phase separation and ribonucleoprotein (RNP) assembly, but traditional methods like in vitro droplet assays and cellular imaging failed to explain how these changes affect RNA binding specificity in vivo. The program tackled this by pursuing five objectives: elucidating RBP functions in stem-cell and neuronal differentiation; exploring multivalent RNA features in RNP condensation; engineering RNA multivalency to manipulate assembly; perturbing IDR-dependent dynamics; and examining species-specific RNP modes.

Key technological breakthroughs included iiCLIP, an enhanced, non-radioactive iCLIP protocol for quantitative RBP-RNA interaction mapping (Lee et al., bioRxiv 2021), and the PEKA computational pipeline for motif discovery and binding region assignment (Kuret et al., Genome Biol, 2022). These tools enabled detailed analyses of RBP mutants across datasets.

Major discoveries emerged from studies on TDP-43, revealing IDR-dependent condensation as crucial for binding long, multivalent RNA regions with dispersed motifs; reduced condensation in mutants caused selective mis-splicing of 3’UTRs and autoregulatory failures (Hallegger et al., Cell, 2021). Collaborative work on UNC13A showed how variants exploit multivalent RNA to evade TDP-43 repression of cryptic exons (Brown et al., Nature, 2022). Further, mapping RBP dynamics during differentiation demonstrated how phosphorylation alters IDR assembly, rewiring mRNA binding and decay (Modic et al., NSMB, 2024).

These findings established multivalent RNA regions as scaffolds gated by IDR dynamics, culminating in the 2025 Nature discovery of "interstasis"—an intermediate state between dispersed binding and full condensation, where RBPs selectively stabilize extended RNA regions without visible aggregates. Interstasis explains how subtle IDR mutations trigger early RNA misregulation in ALS/FTD, shifting paradigms from binary models to finely tuned intermediates vital for splicing, stability, and translation.
Evolutionary analyses of codon biases in multivalent regions illuminated species-specific RNP evolution. Overall, the program’s insights and tools pave the way for therapies targeting IDR-multivalency interactions to correct pathogenic RNA processing, offering hope for selective disease intervention.

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)

The discovery of interstasis revolutionized the understanding of cellular feedback loops, replacing a simplistic binary model with a nuanced mechanistic framework that addresses paradoxes in RNA biology and aging. For over a decade, research on biomolecular condensates portrayed RNA-binding proteins (RBPs) in two states: soluble, diffusely binding RNA locally, or phase-separated into visible condensates. Assessments relied on imaging, in-vitro droplet assays for condensation, and classic CLIP for binding sites. This dichotomy failed to explain how subtle mutations in intrinsically disordered regions (IDRs) trigger selective RNA mis-regulation without apparent aggregates, or why only certain RNAs are affected by altered condensation propensity. Imaging-centric tools lacked sensitivity for detecting intermediate transcriptome-wide assemblies.

Interstasis emerged as an unforeseen intermediate state, where specific RBPs selectively stabilize and organize long, multivalent RNA regions within condensates. Its detection demanded innovative tools: quantitative iiCLIP and comparative iCLIP for precise binding measurements, alongside PEKA algorithms and motif-informed region assignment to quantify multivalent RNA scaffolds. By subtly manipulating IDR proteins and RNA multivalency, we uncovered graded, selective impacts on RNA subsets—insights hidden by prior methods.

This advanced the field as follows:
It found that modest IDR changes (e.g. disease mutations or modifications) disrupt motif-dense regions like 3′UTRs or introns pre-aggregation.
It found that RNA multivalency and IDR propensity predict vulnerable transcripts, enabling testable hypotheses for experiments and therapies.
The methodology offers a versatile blueprint for studying other disease-related RBPs across development or stress.
The work can open new strategies for therapies tuning IDR interactions or RNA scaffolds to restore regulation, bypassing aggregate dissolution.
The work linked sequence motifs to mesoscale behaviors by promoting integrated pipelines (CLIP → PEKA → biophysics → genomics) for RNP analysis.

Ultimately, interstasis reoriented our focus from aggregation to finely tuned IDR–RNA dynamics crafting regulatory micro-states that influence cell fate and disease.

graphical abstract from Hallegger et al, Cell, 2021