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Global characterization of post-transcriptional regulatory interactions in DNA damage response

Final Report Summary - POSTSCRIBEDNADAMAGE (Global characterization of post-transcriptional regulatory interactions in DNA damage response)

1.1. Project objectives
Cellular response to genotoxic stress is a well-characterized network of DNA surveillance pathways. However, the contribution of post-transcriptional regulatory networks to DNA damage response (DDR) has not been extensively studied. PostScribeDNAdamage aimed to identify the protein-RNA interactions during DDR using high-throughput omics approaches. Biochemical, cell, systems biology and computational approaches were employed to study the relevance of post-transcriptional regulators such as RNA-binding proteins (RBPs) during DDR. RBPs are regarded as major trans-acting factors, which regulate gene-expression on post-transcriptional level.
Briefly, the project consisted of 3 major objectives, namely (1) the optimization of experimental conditions to induce DNA damage in vitro, (2) identification of the differential mRNA-bound proteome in response to DNA damage using oligo(dT) purification and mass spectrometry and (3) functional analysis of DDR-specific changes in mRNA-protein interactome (Fig.1).
In addition, the project also proposed to enable a focused career development period in the scope of European mobility for the researcher to gain a high level of expertise in systems biology.

1.2. Description of work
In the initial phase of this project we addressed the feasibility to reproducibly induce DNA damage under our experimental conditions by detection of DNA damage signalling response proteins. Using standard molecular/cell biology techniques, we found that the experimental conditions allow robust induction of DNA damage response even in the presence of perturbations required for the following large-scale experiments (UV exposure and 4-thiouridine treatment). High recovery of mRNA-bound proteins using oligo(dT) purification was ensured. We also found that early time periods after induction of genotoxic stress are most relevant for differences in RBP binding to mRNA, and chose those conditions for further large-scale experiments.
To further address the feasibility of proteomic analyses, we collected several mass spectrometry datasets. Low reproducibility observed in these initial datasets was addressed by modifying our approach to more confidently detect differentially bound proteins between different conditions. The resulting approach based on UV crosslinking, oligo(dT) capture and mass spectrometry (MS) is robust and reproducible. Specifically, spike-in normalization using lysates of cells labelled with stable “heavy” isotope-modified aminoacids (SILAC) was employed to calibrate and accurately quantify the extent of protein binding to mRNA in irradiated and untreated cells. These experiments allowed the identification of almost 200 proteins differentially bound to polyadenylated transcripts upon ionizing irradiation of MCF-7 breast carcinoma cells. Strikingly, most differentially bound proteins showed increased binding to mRNA upon irradiation (N=184). Among them, many known nuclear RNA-binding proteins such as RNA helicases, proteins involved in ribosomal biogenesis and splicing factors were identified, along with known RBPs involved or related to DNA damage response (XRCC6, EWSR1, YBX1, FUS).
We next validated our proteomics datasets by determining differential protein abundances using independent methods and the influence of cellular mRNA abundance on the recovery of mRNA-bound proteins. These data demonstrated the validity and reproducibility of our proteomics datasets.
Finally, we focussed on the functional characterization of several differential mRNA binders, including a DEAD box RNA-helicase that was identified to show highly increased mRNA binding upon IR exposure. We established several stable cell lines with overexpression of FLAG/HA tagged proteins and are in the process of identifying the differential binding sites of proteins using PAR-CLIP as well as their impact on cell proliferation to reveal their mechanism of function in the post-transcriptional regulation of DDR-related phenotypes. In summary, we successfully concluded the work packages 1.1 1.2 2.1 2.2 and are in the process of concluding WPs 3.1 and 3.2.

1.3. Main results
Upon short-term exposure to ionizing irradiation, many RNA-binding proteins bind more abundantly to mRNA. Using our adapted experimental procedure we identified 200 proteins out of approximately 700 detected with increased mRNA binding after IR exposure. Several RNA-binding proteins previously shown to be involved in post-transcriptional regulation during DDR were identified (FUS, EWSR1, XRCC6, YBX1, HNRNPUL2), along with new interesting candidates. Nuclear proteins are overrepresented in this group of proteins, as well as processes such as acetylation and phosphorylation.
We also performed differential protein occupancy profiling on mRNA (Schueler et al., Genome Biol 2014;15:R15.) upon genotoxic stress and identified close to 12.000 potential protein-mRNA contacts that may play a role in the post-transcriptional regulation of DNA damage response. In fact, the transcripts, which showed decreased protein occupancy after IR exposure, are involved in cell cycle related processes, suggesting their important role in DNA damage response. Further work will address the functional and mechanistic of these putative regulatory regions.
During the course of this project I have made significant progress to achieve a great level of expertise in computational approaches, proteomics, high-throughput sequencing, sample preparation and data analysis. I have gained many insights into my work and established several collaborations in the field by attending 2 international conferences on post-transcriptional gene regulation and a workshop on bioinformatics during the course of the project.

1.4. Expected final results and their impact
The final results of PostScribeDNAdamage will contribute to the understanding of factors governing the early post-transcriptional regulatory events during DDR. The main results include a group of candidate proteins, which respond to genotoxic stress by differentially binding to mRNA. As such, they represent novel trans-acting factors, which may play a role in DDR-related cellular processes. One additional benefit of PostScribeDNAdamage is the successful development of a new experimental procedure to identify stress-responsive mRNA-binding proteins on a systems biology level. Due to the recently discovered involvement of RBPs in human diseases ranging from neurologic disorders to cancer, this study is highly relevant for medical research. Further functional analyses of these proteins and their mRNA binding sites will delineate key processes that are involved in post-transcriptional regulation of DNA damage response, impacting such phenotypic responses as cell death, proliferation and differentiation.
In addition, the role of regulatory molecules involved in the de-regulation of DDR in tumours is critical to explore new targets in the DDR pathway for designing novel anticancer therapies. Recovery of functional DDR responses such as cell cycle and growth arrest in tumours may be a powerful and selective way to target tumour cells. Discovery of new markers which define differences in post-transcriptional regulation of DDR may be eventually used in diagnostics and pharmacogenetic testing and consequently lead to rationalization of existing therapies. This may represent a long-term benefit for the society in the form of increased quality of life and reduction of health care costs.
In summary, the obtained knowledge implemented by PostScribeDNAdamage is scientifically significant due to the originality of obtained results as well as their potential in translational medicine in the form of novel candidates, which modulate DDR-related pathologies such as cancer and neurodegenerative disorders.

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