Studying RNA-binding proteins involved in neural differentiation and degeneration

Project Information

NeuroRBP

Grant agreement ID: 799948

DOI

10.3030/799948

Project closed

EC signature date 4 April 2018

Start date 1 June 2019

End date 31 May 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 195 454,80

EU contribution

€ 195 454,80

Coordinated by

THE FRANCIS CRICK INSTITUTE LIMITED
United Kingdom

Periodic Reporting for period 1 - NeuroRBP (Studying RNA-binding proteins involved in neural differentiation and degeneration)

Reporting period: 2019-06-01 to 2021-05-31

Summary of the context and overall objectives of the project

RNA-binding proteins (RBPs) regulate multiple levels of gene expression. Furthermore, RBPs support biomolecular condensate development, and contribute to the functionality of enhancers, transcription factors and RNA Pol II. Mutations and dysregulated expression of RBPs may lead to neurodegeneration. The main question of this project is why are neurons very sensitive to the malfunction of RBPs? And how does this malfunction end up to neurodegenerative disorders?
At the moment, there is no successful therapy to treat devastating symptoms of neurodegenerative disorders. Considering the sever demand, there are extensive efforts to develop novel therapeutic approaches. For Europe, as a research-based society and economy, these studies are crucial to achieve a leading global position in the field of molecular biology. This project helps to understand the pathways involved in neurodegenerative diseases from chromatin and RBP perspectives, and to develop more effective treatments.
In this study we aimed to develop a highly sensitive method to study dynamics of chromatin-binding in RBPs. We used the method to compare motor neurons obtained from patient-derived iPS cells with control cells. As a result, we shed light on the mechanism that makes the motor neurons vulnerable to DNA damage.

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

Initially, to improve protein identification by mass spectrometry I developed an approach to chemically modify streptavidin beads (protease-resistant streptavidin). In fact, purification of biomolecules via streptavidin is extensively utilized in molecular biology and biochemistry to identify biotinylated biomolecules and their interaction partners. However intense streptavidin-derived peptides affect protein identification by mass spectrometry. My approach for chemical modification of streptavidin reduces the contaminations over 100-fold. As a result, proteins interacting with diverse biotinylated bait-molecules such as DNA, proteins and lipids are identified with deeper coverage. The results were published in a peer-reviewed journal (Rafiee MR et al. MSB 2020).

After that I developed SPACE (Silica Particle Assisted Chromatin Enrichment), a straightforward and highly sensitive method for isolation of chromatin and identification of RBP using mass spectrometry. I evaluated SPACE by studying the global chromatin composition of mES cells. I successfully identified previously reported DNA- and chromatin-binding proteins, as well as many RBPs. Surprisingly, RBPs comprise ~48% of the proteins obtained from the chromatin composition. To understand how RBPs bind to chromatin, I developed SPACEmap which indicates that intrinsically disordered regions (IDRs) are frequently employed by chromatin proteins, including chromatin-associated RBPs, for chromatin-binding. I also took advantage of protease-resistant streptavidin to invent SPACE-SICAP, a more stringent version of SPACE which confirms previous findings with higher confidence. Altogether, I demonstrated various applications of SPACE and provided a systematic view of RBP-chromatin interactions. The results were submitted to a peer-reviewed journal, and is available in bioRxiv (https://doi.org/10.1101/2020.07.13.200212)
I then harnessed the power of SPACE to interrogate chromatin proteome composition in neuronal differentiated from ALS-patient-induced pluripotent stem (iPS) cells. I find that mutant VCP has diminished chromatin-binding, which is associated with chromatin release of several DNA damage response factors, such as TP53BP1. Conversely, I observed increased chromatin recruitment of RNA quality control pathways in the mutant cells. These data highlight the critical function of VCP in promoting DNA repair such that ALS-causative VCP mutations make cells vulnerable to DNA damage. Currently, I am preparing a manuscript to publish the results.

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)

Additionally, I sought to understand how VCP mutations modify the proteome associated with RNA polymerase II (Pol II) in patient-derived iPS cells undergoing motor neurogenesis. Remarkably, I observed that several RNA processing components bound to Pol II are downregulated in patient cells. The question is how downregulation of the RBPs affects RNA processing. My approach thus provides a mechanistic insight into the perturbed transcriptional processes in VCP-associated ALS. I hope my findings will improve our understanding of the disease processes and will eventually contribute to the disease treatment.

mutation in RBPs makes neurons vulnerable to DNA damage