Angiogenin-induced tRNA-derived Stress-induced RNAs Promote Stress-induced Stress Granule Assembly

Paul Anderson,Pavel Ivanov,Guo-Fu Hu,Nemisha Dawra,Nancy Kedersha,Mohamed M. Emara,Tyler Hickman,Sarah Tisdale

doi:10.1074/jbc.m109.077560

Paul Anderson, Pavel Ivanov + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.077560

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Abstract

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 (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate 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.

Highlights

Grants AI065858, AI033600, and AR0514732. □S The on-line version of this article contains supplemental Figs. 1–5. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: Division of Rheumatology, Immunology and Allergy; Brigham and Women’s Hospital, Harvard Medical School, Smith Bldg. 652, One Jimmy Fund Way, Boston, MA 02115
We previously showed that phosphoryla- either control FLAG-LSM1 or FLAG-RNH1 constructs prior for tRNA-derived stress-induced RNAs (tiRNAs)-induced translational repression [4]
This reprogramming is initiated by phospho-eIF2␣-induced translational repression and facilitated by active sequestration of untranslated mRNAs into stress granules (SGs)

Summary

EXPERIMENTAL PROCEDURES

Tissue Culture and Cell Treatments—Parental U2OS cells as well as stable transfectants expressing recombinant proteins were maintained at 37 °C in a CO2 incubator in minimal essential medium supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin/streptomycin (Sigma). After 30 min, fresh metabolic labeling medium containing ϳ200 ␮Ci of L-[35S]methionine (EasyTag Express 35S Protein Labeling Mix; PerkinElmer Life Sciences) and wild type or P112L mutant ANG (0.5 ␮g/ml) in the presence or absence of 70 ␮M sodium arsenite or 15 nM pateamine A was added to each well, and cells were incubated at 37 °C for 1 h. Assembly—We previously reported that transfection of 5Ј- but not 3ЈANG-induced natural tiRNAs inhibits protein synthesis in U2OS cells [4] To determine whether this erslips were mounted in polyvinyl mounting medium, and result correlates with the assembly of SGs, we transfected the cells were viewed and photographed with an Eclipse E800 U2OS cells with natural 5Ј-tiRNAs (Fig. 2C) or 3Ј-tiRNAs fluorescence microscope (Nikon) equipped with a digital (Fig. 2B) or control RNAs (Fig. 2A; 750 nM final concentracamera (CCD-SPOT RT, Diagnostic Instruments) using a tions) using Lipofectamine 2000. The images were merged and revealed that optimal induction of SGs occurred 7 h after analyzed using Adobe Photoshop (version 10)

RESULTS

Findings

DISCUSSION