NEAT1 Modulates the TIRR/53BP1 Complex to Maintain Genome Integrity

Tudor Interacting Repair Regulator (TIRR) is an RNA-binding protein (RBP) that interacts directly with 53BP1, restricting its access to DNA double-strand breaks (DSBs) and its association with p53. We utilized iCLIP to identify RNAs that directly bind to TIRR within cells, identifying NEAT1_1, the short isoform of the long noncoding RNA NEAT1, as the primary RNA partner. The high affinity of TIRR for NEAT1_1 is due to prevalent G-rich motifs in NEAT1_1. This interaction destabilizes the TIRR/53BP1 complex, promoting 53BP1's function. NEAT1_1 is enriched during the G1 phase of the cell cycle, thereby ensuring that TIRR-dependent inhibition of 53BP1’s function is cell cycle-dependent. TDP-43, an RBP that is implicated in neurodegenerative diseases, modulates the TIRR/53BP1 complex by promoting NEAT1_1 production. Together, we infer that NEAT1_1, and factors regulating NEAT1_1, may impact 53BP1-dependent DNA repair processes, with implications for a spectrum of diseases. iCLIP was used to identify RNA bound by FLAG-tagged TIRR protein. A comparison was made between untreated cells and cells irradiated with 5Gy. Briefly, U2OS cells stably transfected with a Flag version of TIRR, or EV, were UV-crosslinked on ice and lysed in RIPA buffer. For the irradiation experiment, cells were irradiated at 5 Gy with an X-ray source. Two replicates were prepared for each condition. 0.4 Units of RNaseI and 4 Units Turbo DNase were added per 1 mL of cell lysate at 1 mg/mL protein concentration for RNA fragmentation. Antibodies against the Flag coupled to magnetic Protein G beads were used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labeled IRL3 adaptor with the following sequence: /5rApp/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA /iAzideN/AA AAA AAA AAA A/3Bio/. For full experimental protocol see: Lee, F. C. Y. et al. An improved iCLIP protocol. bioRxiv, 2021.2008.2027.457890 (2021). https://doi.org/10.1101/2021.08.27.457890 RNA_seq: To identify genes whose expression is impacted by gamma radiation in osteosarcoma cell line U2OS