Dissecting the molecular mechanisms underlying hnRNP exclusion from DNA double-strand breaks.

Informazioni relative al progetto

hnRNPs-DNA damage

ID dell’accordo di sovvenzione: 101150643

DOI

10.3030/101150643

Progetto chiuso

Data della firma CE 14 Marzo 2024

Data di avvio 1 Aprile 2024

Data di completamento 31 Marzo 2026

Finanziato da

• Marie Skłodowska-Curie Actions (MSCA)

Costo totale

Nessun dato

Contributo UE

€ 173 847,36

Coordinato da

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - hnRNPs-DNA damage (Dissecting the molecular mechanisms underlying hnRNP exclusion from DNA double-strand breaks.)

Periodo di rendicontazione: 2024-04-01 al 2026-03-31

Sintesi del contesto e degli obiettivi generali del progetto

DNA double-strand breaks (DSBs) are highly toxic lesions that must be efficiently repaired to maintain genome stability. Cells react to DSBs by activating a complex signalling network termed the DSB response which senses, signals and repairs DSBs. Emerging organizers of this pathway are various RNAs and RNA-binding proteins (RBPs). Yet, our knowledge on how these RNAs and RBPs participate in the DSB response and how they are regulated is largely incomplete.

Our laboratory developed a targeted microscopy screen to investigate the mobility of a RBP family called heterogeneous nuclear ribonucleoproteins (hnRNPs) at DSB sites. Work from our lab and other labs have shown that many hnRNPs are actively excluded from damaged chromatin soon after the induction of DSBs. However, the mechanism and significance of this exclusion are still unexplored. This project fills this important gap by providing a detailed knowledge of how hnRNPs are integrated into the DSB response.

Using cutting-edge imaging technologies, we propose to characterize the exclusion properties of hnRNPs at DSBs and to understand the significance of hnRNP exclusion for DNA repair (Aim 1). We also aim to identify the local protein and RNA complexes associated with hnRNPs at DSB sites using proximity labelling proteomics and RNA-sequencing approaches (Aim 2).

Overall, our results show that the exclusion of hnRNPs to DSBs participates in the coordination between RNA biology and the DSB response and is essential to facilitate a timely and accurate DSB repair.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

We first successfully set up laser microirradiation and subsequent time-lapse imaging with a new spinning disk confocal microscope (available at the in-house microscopy core facility). Using this system, we characterized the relative mobility of the entire hnRNP family at DSBs, both in space and time. We then proceeded to identify the determinants of hnRNP exclusion. This end, we assessed whether inhibition of key components of early, medium and late DSB response affects hnRNP localization to laser microirradiation-induced DSBs, also studied the impact of transcription activation and blockage on hnRNPs mobility at DSBs, and mapped relevant domains on a model hnRNP factor. Together, the results from these experiments provided new insights into the signalling mechanisms that coordinate hnRNPs exclusion at DNA damage sites.

In addition, we also performed experiments to assess the functional significance of hnRNP exclusion for DNA repair. To this end, we generated hnRNP transgenes that can be stably tethered to DNA breaks and assessed the impact of hnRNP tethering on DSB signalling and repair. Our results showed that the exclusion of hnRNPs from damaged chromatin is essential for proper activation of the DSB response.

To identify the protein complexes associated with excluded hnRNPs at DSB sites, we made use of the TurboID system, a technology based on the expression of a fusion between a bait protein (here hnRNPs) and the E.coli biotin biotin ligase mutant TurboID. In the presence of biotin, this mutant biotinylates proteins that are near neighbours of the fusion proteins (about within a radius of 10 nm) thus allowing for the efficient isolation of biotinylated proteins and subsequent identification by mass spectrometry (MS). To date, we have generated HEK293 cells expressing TurboID-hnRNPF and performed MS analysis. We are currently extending this MS setup to other hnRNP family members. Ultimately, we anticipate that the MS results will be critical to decipher how the hnRNPs interact with each other and with other proteins at DSBs, and how the different complexes are integrated into the overall DSB response.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

We have shown that the laser microirradiation strategy is well suited to characterizing and studying hnRNPs functions in the context of DNA damage. This enabled us to better understand the timing of RNA-related events at damaged chromatin and how RBPs are integrated in the DSB response. Specifically, our findings elucidated the mechanisms by which hnRNPs are excluded from damaged chromatin. In addition, we demonstrated that hnRNPs exclusion is essential for the recruitment of DSB signalling factors to damaged-chromatin. By highlighting the biological significance of hnRNPs exclusion in DSB response activation, this project broadens the scope of genome maintenance mechanisms.

Further work focusing on the identification of the local protein and RNA complexes associated with hnRNPs at DSB sites will enable us to go well beyond the current knowledge of the field to characterize novel factors that coordinate RNA biology at DSB sites.

Outline of the project