mirnaAGOddr · Regulation of Argonaute-2 and miRNA-dependent mRNA decay in response to oxidative stress and DNA damage

Regulation of Argonaute-2 and miRNA-dependent mRNA decay in response to oxidative stress and DNA damage.

Maintenance of genome stability through many cell generations requires the precise duplication of DNA in S phase, the timely repair of DNA lesions and the faithful segregation of chromosomes during mitosis. Defects in these processes result in genomic instability, a hallmark of cancer important for the initiation and progression of tumors (1). The RNA-binding proteins TIA1 and TIAR (TIAL1) play important roles in cell survival, proliferation and stress responses (2). The two proteins preferentially bind to U-, CU- or AU-rich RNA sequences (3), but also have affinity towards ssDNA (4). TIA1 and TIAR are nucleo-cytoplasmic shuttling proteins that regulate alternative splicing. Under adverse conditions such as heat shock or oxidative stress, TIA1 and TIAR accumulate in the cytoplasm where they act as repressors of translation (5,6). Interestingly, the homozygous deletion of TIAR in mice causes severe embryonic lethality, and surviving TIAR-/- mice are infertile due to loss of primordial germ cells (7). In cell culture models, TIA1 and TIAR share characteristics with tumor suppressor genes: their overexpression promotes apoptosis, while depletion enhances cell proliferation and growth of xenograft tumors (8,9). In accordance with a tumor suppressive role, TIA1 and TIAR protein levels are reduced in human tumors of the lung, skin, breast, colon, uterus, pancreas and brain (8). These studies, however, did not link TIAR expression to cancer patient prognosis, nor did they uncover the cellular mechanism by which TIAR controls cell proliferation. Since, it was recently described that RNA-splicing components were essential to maintain genomic stability our objectives were to characterizes the role of TIAR during the cell cycle and in response to DNA Damage. For these we proposed the next objectives: 1) Analysis of TIAR expression in head and neck squamous cell carcinoma. 2) Analysis of cell cycle checkpoint in TIAR kd cells. 3) Study the role of TIAR in response to replication stress. 4) Study the role of TIAR in response to DNA damage. Our results demonstrate that TIAR is an essential component of the S-M checkpoint required for genome stability. Knockdown of TIAR sensitizes cells to replication stress and ATR inhibition, causes hyperphosphorylation of histone H3 and leads premature mitotic entry. As a consequence, TIAR-depleted cells acquire chromosomal aberrations and undergo mitotic catastrophe. Moreover, we discovered that replication stress causes TIAR, RNA polymerase II, and core splicing factors to accumulate in novel, prophase-specific subnuclear structures termed replication stress bodies (RSBs). Co-localization of RPA, RNase H1 and FANCD2 reveals RSBs as sites where active transcription and stalled replication persist during prophase. Finally, we found that TIAR acts independently of p53, and low TIAR expression in squamous cell carcinoma to correlate with reduced patient survival, demonstrating that TIAR has tumor suppressive activity. Our results uncover TIAR as a factor that coordinates transcription with DNA replication at the entry of mitosis and thereby exerts an essential function in maintenance of genome stability. Our results also demonstrate that low TIAR expression levels in head and neck squamous cell carcinoma (HNSCC) are associated with poor patient survival. In normal mucosa, TIAR was detected predominantly in the cytoplasm of supra-basal keratinocytes, while a more heterogeneous staining pattern, ranging from low to high TIAR levels, was observed in tumor tissues. For all patients (n=74) who had received radiotherapy, the subgroups with high and low TIAR levels were compared. Interestingly, TIAR expression did not correlate with age, gender, tumor size, lymph node metastasis, tumor grade, tobacco and alcohol consumption or HPV infection status and univariate Kaplan-Meier analysis revealed that TIARlow levels represent an unfavorable risk factor for both progression-free (p = 0.033) and overall survival (p = 0.038) in HNSCC patients, indicating that TIAR act as a tumor suppressor in HNSCC. Our results demonstrate that TIAR down-regulation could be used as a prediction marker for tumor malignancy. At the cellular level, we found that depletion of TIAR sensitizes cells to the ATR inhibitor ATRi. Recently, ATR inhibitors are being used to clinical trial to specifically kill cancer cells with high levels of replicative stress. This is the case of knock cell for p53 or cells overexpressing Cyclin D. Then, since low TIAR induce replication stress, we propose that ATRi can be used in patients with low levels of TIAR. 1. Gordon, D.J., Resio, B. & Pellman, D. Causes and consequences of aneuploidy in cancer. Nat Rev Genet 13, 189-203 (2012). 2. Anderson, P. & Kedersha, N. Stress granules: the Tao of RNA triage. Trends Biochem. Sci. 33, 141-50 (2008). 3. Ivanov, P., Kedersha, N. & Anderson, P. Stress puts TIA on TOP. Genes Dev 25, 2119-24 (2011). 4. Suswam, E.A., Li, Y.Y., Mahtani, H. & King, P.H. Novel DNA-binding properties of the RNA-binding protein TIAR. Nucleic Acids Res 33, 4507-18 (2005). 5. Mazan-Mamczarz, K., Lal, A., Martindale, J.L., Kawai, T. & Gorospe, M. Translational repression by RNA-binding protein TIAR. Mol Cell Biol 26, 2716-27 (2006). 6. Damgaard, C.K. & Lykke-Andersen, J. Translational coregulation of 5'TOP mRNAs by TIA-1 and TIAR. Genes Dev 25, 2057-68 (2011). 7. Beck, A.R., Miller, I.J., Anderson, P. & Streuli, M. RNA-binding protein TIAR is essential for primordial germ cell development. Proc Natl Acad Sci U S A 95, 2331-6. (1998). 8. Izquierdo, J.M., Alcalde, J., Carrascoso, I., Reyes, R. & Ludena, M.D. Knockdown of T-cell intracellular antigens triggers cell proliferation, invasion and tumour growth. Biochem J 435, 337-44 (2011). 9. David, C.J. & Manley, J.L. Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged. Genes Dev 24, 2343-64 (2010).

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