FORTH (G. Garinis group): NER and RNA splicing in development and disease
i. Objective of research: To identify XAB2 (XPA-binding protein 2)-associated protein complexes and RNA targets in murine development.
ii. Current state of the art: Besides skin cancer or aging, mutations in NER genes may trigger developmental defects as seen in NER disorders. Recent studies have shown that distinct NER factors play a role in transcription and certain NER proteins are recruited to active promoters in vivo.. XAB2, containing tetratricopeptide repeats, was recently shown to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 binds Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and our preliminary data show that it is involved in RNA splicing, the DDR and transcription. At present, however, no biochemical in vivo evidence exists as to whether and how XAB2 is involved in such fundamental mechanisms, what are the underlying multi-protein complexes or how XAB2 coordinates and prioritizes its distinct roles in RNA splicing and DNA repair during development and disease. Here, we aim to provide insights into the functional role of XAB2 in development and disease in vivo.
iii. Research methodology and approach: We will focus on the identification of XAB2-bound protein complexes and RNA targets in mice. To do this, we will use an in vivo biotinylation tagging methodology for the efficient direct purification of XAB2 protein partners and established RNA immunoprecipitation approaches (RIP) to identify the relevant RNA targets upon DNA damage, upon transcription stimulation and during mammalian development. More specifically, XAB2 will be tagged by fusing the 14aa biotinylation tag to XAB2 protein and expressing the biotinylation tag-fused XAB2 protein in established cell lines expressing the BirA biotin ligase knocked-in the Rosa26 locus. BirA specifically recognizes and biotinylates the short tag, creating a very high affinity “handle” for isolating tagged factors by binding to streptavidin. We will generate biotin-tagged XAB2 knock-in mice in the BirA genetic background by fusing the 14aa biotinylation tag to the endogenous C terminus of XAB2. This will be used to specifically tag XAB2 for mass spectrometry and for RIP-coupled massive parallel sequencing (MPS) approaches in tissue protein extracts derived from bio-XAB2 knock-in mice; as Xab2-/- mice are lethal, the bioXAB2 animals also carry loxP sites allowing the cell type-specific ablation of XAB2.
iv. Originality and innovative aspects of the ESR project: Using a unique series of biotin-tagged NER mice, we propose to dissect the functional contribution of XAB2 in vivo gaining insights into the functional role of NER in RNA splicing, NER disorders and age-related diseases.
v. Integration of the ESR project to the overall research programme: Our ESR will work with the Schumacher group to dissect the functional relevance of identified protein complexes in C. elegans screening assays and with Norgenotech and LXRepair on the development of high throughput screening assays for the detection of DSBs in in NER-defective animals.