Double strand break repair protein TIRR regulates RNA metabolism in response to DNA damage and cell stress [RNA-Seq]
Tudor interacting repair regulator (TIRR) is a well-studied p53 binding protein 1 (53BP1) interactor in response to DNA double strand breaks (DSBs). TIRR is also an RNA binding protein, however its role in RNA regulation in response to cellular insults is not understood.  Here we show that TIRR binds to specific group of mRNAs in response to DNA damage. TIRR bound transcripts encode for transcription factors and RNA polymerase II (RNAPII) transcription regulators. Furthermore, TIRR modulates the formation of RNA processing bodies (P bodies/PBs), in response to DSBs. TIRR also regulates the formation of the closely related but distinct compartments known as stress granules (SGs) in response to cell stress. Furthermore, TIRR dissociates from 53BP1 in response to sodium arsenite treatment independently of Ataxia Telangiectasia Mutated Protein (ATM) signalling. Finally, TIRR interacts with the nuclear export protein Exportin-1 (XPO1) upon DNA damage. TIRR bound RNA co-localises with PB and SGs and TIRR depletion leads to nuclear RNA retention. This work reveals that TIRR orchestrates mRNA nuclear export, metabolism and storage in PBs/SGs. Hence TIRR regulates RNA mediated DNA damage and cell stress responses independently of its canonical 53BP1 binding role. Overall design: RNA immunoprecipitation and sequencing (RIP-Seq) experiments were perfomed to identify specific RNAs bound to TIRR after DNA damage using TIRR-GFP and GFP cells.  Hela cells with integrated inducible TIRR-GFP or GFP only (as a control for background) were used to induce expression of TIRR-GFP or GFP with doxycycline. DNA double strand breaks were induced with Etoposide (Eto). Total RNA-sequencing was performed in stably integrated inducible shRNA Hela cells, expressing shRNA for TIRR or GFP as a control. TIRR knockdown was induced using doxycycline for 48 hours. RNA was collected before andafter DNA damage,inducedby IR and followed by 1 hour incubation,or by ETO incubation for 2 hours. It was thensubjected to paired-end total RNA sequencing on the Illumina NovaSeq6000.