PROJET RNAcan!

Scaffolding RNAs in cancer biology

Ce projet est porté par le CNRS DR Occitanie-Est – Institut de génomique humaine (IGH).

CONTEXT

RNA therapeutics in cancer

RNA has held long-standing hopes for human medicine and the recent implementation of RNA vaccines has demonstrated their incredible potential in a spectacular manner. The labile nature of RNAs, their lack of integration in the genome and their poor immunogenicity make them safe, while their versatility as coding and non-coding molecules opens their use to virtually any disease. RNAs can be used as biomarkers to detect a disease, as targets to alter a process or an ‘undruggable’ protein, or directly as a therapeutic molecules. In this project, we concentrate on an entirely new function of RNA molecules in scaffolding large molecular assemblies driving cell compartmentalization. In the last few years, the formation of molecular condensates has emerged as a fundamental mechanism for cell compartmentalization and consequently, for cell life. Biological condensates form by phase separation involving demixing, i.e. the formation of a liquid droplet separated from the cellular milieu. This process is driven via multivalent interactions between the droplet components, which allows their aggregation when their concentration reaches a critical threshold. An important feature of various condensates is that RNA plays an essential role in their formation. Here, we propose that RNAs could play a general role in cellular compartmentalization by controlling the formation of many cellular condensates. We are testing this new vision of RNA molecules as general cellular scaffolders.

OBJECTIVES

Starting from the hypothesis that RNAs are general scaffolding molecules that drive cell compartmentalization at a scale unsuspected before, our project aims to demonstrate this paradigm shifting hypothesis and determine how aberrant RNA-driven cell compartmentalization leads to cancer. We have recently discovered several large molecular assemblies for which we suspect an essential role of RNAs in their formation. These assemblies function in DNA repair, splicing and translation, three cellular processes that play major roles in cancer.

Taking advantage of these three cellular processes in which the scaffolding role of RNA is emerging, we aim to unravel the general functional principles of RNA-driven compartmentalization, following four specific objectives:

1. To identify new RNA-based compartments and scaffolding RNAs;
2. To decipher their roles in DNA repair, splicing and translation;
3. To characterize the mechanisms of RNA-driven compartmentation;
4. To unravel the roles of scaffolding RNAs during cancer progression.

This project will provide a new vision of the role of RNAs in cancer biology and open new possibilities to fight cancer.

RESULTS

DNA repair: We showed that the Fanconi anemia protein SLX4 forms globular clusters of condensates on chromatin that compartmentalize DNA damage response proteins. Compartmentalization of the SUMO/RNF4 pathway by SLX4 triggers the extraction of topoisomerase 1-DNA-protein cross-links from chromatin and the degradation of nascent DNA (1, 2). Investigations into the role of RNA in the scaffolding of SLX4 condensates are underway.

Splicing: 11% of the alternatively spliced genes regulated during the epithelial-to-mesenchymal transition are differentially enriched in H3K27ac and CTCF, which are two hallmarks of enhancer sequences. Using chromosome conformational capture assays (4C-seq and microC), we have uncovered a 3D higher-order genome organization in which alternatively spliced exons physically interact with long distant regions important for the final splicing outcome.

Translation: Germ granules are specific RNA granules that contain »200 mRNAs and specify the germline. Using these granules as a model of cytoplasmic RNA granules we found that they are biphasic -composed of two immiscible phases- and that translation occurs in the outer phase, while translational repression takes place in the internal phase. Moreover, RNA recruitment to the granules is required for their biphasic architecture. These results reveal a key role of RNA and the biphasic organization in the compartmentalization of RNA granule functions (3).

In another study we analyzed the key oncogene b-catenin that is regulated post-translationally by the destruction complex composed of Axin and the tumor suppressor APC. Using RNA immunoprecipitation and advanced microscopy, we found that the destruction complex acts co-translationally and that co-translational interactions take place in dedicated compartments formed by the condensation of b-catenin polysomes by the destruction complex. Interestingly, inhibition of these co-translational interactions prevents the tumor suppressing activity of the destruction complex towards b-catenin.

RECENT PUBLICATIONS

1. Alghoul E, Paloni M, Takedachi A, Urbach S, Barducci A, Gaillard P-H, Basbous J, Constantinou A. Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair. Molecular Cell. 2023; 83(10):1640-58. doi: 10.1016/j.molcel.2023.03.021. PubMed PMID: 37059091

2. Alghoul E, Basbous J, Constantinou A. Compartmentalization of the DNA damage response: Mechanisms and functions. DNA Repair. 2023; 128:103524. doi: 10.1016/j.dnarep.2023.103524. PubMed PMID: 37320957

3. Ramat A, Haidar A, Garret C, Simonelig M. Germ granule higher-order organization coordinates their different functions. bioRxiv 2023. doi: https://doi.org/10.1101/2023.11.24.568558

4.Bellec M, Chen R, Dhayni J, Favard C, Trullo A, Lenden-Hasse H, Lehmann R, Bertrand E, Lagha M, Dufourt J. Boosting the toolbox for live imaging of translation. bioRxiv 2023. doi: https://doi.org/10.1101/2023.02.25.529998