Double strand break repair protein TIRR regulates RNA metabolism in response to DNA damage and cell stress [RNA-Seq]. 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.

Tudor相互作用修复调控因子（Tudor interacting repair regulator, TIRR）是被广泛研究的p53结合蛋白1（p53 binding protein 1, 53BP1）的相互作用伙伴，参与DNA双链断裂（DNA double strand breaks, DSBs）应答反应。TIRR同时也是一种RNA结合蛋白，但目前对于其在细胞应激应答中参与RNA调控的功能仍不明确。本研究发现，TIRR可在DNA损伤应答过程中结合特定群组的信使RNA（messenger RNA, mRNA）。TIRR结合的转录本编码转录因子及RNA聚合酶II（RNA polymerase II, RNAPII）转录调控因子。此外，TIRR可调控DNA双链断裂应答过程中RNA加工小体（RNA processing bodies, P bodies/PBs）的形成。同时，TIRR还可调控细胞应激应答中一类与RNA加工小体密切相关但功能独立的无膜细胞器——应激颗粒（stress granules, SGs）的形成。此外，在亚砷酸钠处理诱导的应激中，TIRR可脱离53BP1，且这一过程不依赖于共济失调毛细血管扩张突变蛋白（Ataxia Telangiectasia Mutated Protein, ATM）信号通路。最后，DNA损伤时TIRR可与核输出蛋白Exportin-1（XPO1）发生相互作用。TIRR结合的RNA可与RNA加工小体及应激颗粒共定位，且TIRR敲低会导致核内RNA滞留。本研究揭示，TIRR可调控信使RNA的核输出、代谢以及在RNA加工小体/应激颗粒中的储存过程。因此，TIRR可独立于其经典的53BP1结合功能，调控RNA介导的DNA损伤与细胞应激应答过程。实验整体设计：本研究通过RNA免疫共沉淀测序（RNA immunoprecipitation and sequencing, RIP-Seq）实验，利用表达TIRR-绿色荧光蛋白（TIRR-GFP）与仅表达绿色荧光蛋白（green fluorescent protein, GFP）的细胞系，鉴定DNA损伤后与TIRR结合的特异性RNA。实验采用整合了可诱导型TIRR-GFP或仅GFP（作为背景对照）的HeLa细胞系，通过多西环素（doxycycline）诱导TIRR-GFP或GFP的表达。采用依托泊苷（Etoposide, Eto）诱导DNA双链断裂。同时，本研究在稳定整合了可诱导型短发夹RNA（short hairpin RNA, shRNA）的HeLa细胞系中开展总RNA测序，该细胞系可表达靶向TIRR的shRNA或作为对照的靶向GFP的shRNA。通过多西环素诱导TIRR敲低，诱导时长为48小时。分别在DNA损伤处理前及处理后收集RNA：DNA损伤可通过电离辐射（ionizing radiation, IR）诱导，诱导后孵育1小时；或通过依托泊苷处理2小时诱导。随后将提取的RNA在Illumina NovaSeq6000测序平台上开展双端总RNA测序。