1 ANGIOGENIN-INDUCED tiRNAs PROMOTE STRESS-INDUCED STRESS GRANULE ASSEMBLY Mohamed M. Emara 1,2 *, Pavel Ivanov 1 *, Tyler Hickman 1 , Nemisha Dawra 1 , Sarah Tisdale 1 , Nancy Kedersha 1 , Guo-Fu Hu 3 , Paul Anderson 1 1- Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, 02115 2- Department of Virology, School of Veterinary Medicine, Cairo University, Giza, Egypt, 12211 3- Department of Pathology, Harvard Medical School, Boston, Massachusetts, 02115 *-these authors contributed equally to this manuscript Running title: tiRNAs promote stress granule assembly Address Correspondence to: Paul Anderson, MD PhD Division of Rheumatology, Immunology and Allergy; Brigham and Women's Hospital, Harvard Medical School; Smith Building 652; One Jimmy Fund Way, Boston, MA 02115 Phone: 617-525-1202 Fax: 617-525-1310 Email: panderson@rics.bwh.harvard.edu Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'-, but not 3'-, tRNA fragments (tiRNAs) induces the phospho-eIF2α-independent assembly of SGs. Natural 5'-, but not 3'-, tiRNAs are capped with a 5' mono-phosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG and tiRNA-induced stress response program. In response to environmental stress, eukaryotic cells activate stress-response programs that down-regulate energy-expensive processes such as transcription and translation. These regulatory programs reduce the expression of common housekeeping genes, while increasing the expression of genes that repair stress-induced damage and promote cell survival. At the level of translation, this is achieved by exploiting the differential sensitivity of mRNAs to changes in the availability or activity of general initiation factors such as eIF2α (1). Phosphorylation of eIF2α by one of several stress-activated kinases reduces the availability of the eIF2-GTP- tRNA i Met ternary complex to inhibit translation initiation. This reduces the translation of most transcripts but enhances the translation of transcripts possessing regulatory upstream open reading frames such as transcription factor ATF4, a component of the integrated stress response program. Thus, phospho-eIF2α triggers a profound re-programming of cellular protein synthesis that helps cells adapt to adverse environmental conditions. Stress-induced cleavage of tRNA initiates a complementary stress response program found in both prokaryotes and eukaryotes (2). In mammals, stress-induced tRNA cleavage is mediated by ANG (ANG) (3,4), a 14 kDa member of the pancreatic ribonuclease A (RNAse) super-family. ANG is a secreted endoribonuclease that possesses both angiogenic (5) and cytoprotective activities (6,7). Secreted ANG enters cells via receptor-mediated endocytosis (8,9), translocates to the nucleus (10), and promotes ribosomal RNA transcription and cellular proliferation (11-13). ANG secretion is stimulated by hypoxia, suggesting that it may serve as a stress-induced paracrine factor that protects neighboring cells from deleterious effects of stress. In a previous study, we showed that stress promotes ANG-mediated tRNA cleavage to produce tRNA-derived stress-induced RNAs (tiRNAs) (4). Cleavage occurs preferentially in the anticodon loop of mature tRNA to produce 5' and 3' fragments (5’ and 3’ tiRNAs, respectively). Addition of recombinant wild-type, but not RNAse-inactive mutant, ANG to cultured cells promotes tiRNA production and inhibition of protein synthesis. Thus the ribonuclease activity http://www.jbc.org/cgi/doi/10.1074/jbc.M109.077560 The latest version is at JBC Papers in Press. Published on February 3, 2010 as Manuscript M109.077560 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from by guest on May 12, 2016 http://www.jbc.org/ Downloaded from