FEBS Letters 588 (2014) 3454–3460
journal homepage: www.FEBSLetters.org
mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress Kazunori Watanabe a,b,⇑, Kenichi Ijiri b, Takashi Ohtsuki a a b
Department of Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan Radioisotope Center, The University of Tokyo, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 18 June 2014 Revised 4 August 2014 Accepted 4 August 2014 Available online 12 August 2014 Edited by Michael Ibba Keywords: Mammalian target of rapamycin tRNA degradation Xrn2 Heat stress
a b s t r a c t Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNAMet (iMet) through 50 –30 exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Eukaryotic cells possess mechanisms that respond to environmental stresses, including heat, oxidation, and nutrient starvation. The major stress-response mechanisms include DNA damage repair, cell-cycle arrest, and translational suppression [1,2]. The upregulation of heat shock factors, modification of specific proteins, formation of stress granules (SGs) and nuclear stress bodies, and modulation of RNA metabolism and translocation are important components of the stress response [1–4]. Transfer RNA (tRNA) is a fundamental component of the translation machinery that plays a key role in modulating translation during cellular stress. For instance, non-methionine-tRNAs (e.g., tRNALeu) are aminoacylated with methionine in HeLa cells under conditions of oxidative stress. The subsequent increase in mischarged tRNAs leads to the misincorporation of methionine during protein synthesis [5]. Moreover, tRNA fragments are produced by the endonuclease Rny1 in response to nutrient starvation and oxidative stress in yeast [6,7]. In mammalian cells under oxidative stress, tRNA fragments (tiRNAs) generated by angiogenin (ANG) endonuclease suppress global protein translation [8–10]. In addition, tiRNAs trigger the assembly of stress granules, which transiently store untranslated mRNA–protein complexes in U2OS ⇑ Corresponding author at: Department of Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan. E-mail address: [email protected] (K. Watanabe).
cells [11]. The formation of SGs is an important mechanism by which cells reprogram translation to survive in adverse conditions [12]. The activation of Rny1 in yeast and ANG in mammalian cells is important for oxidative stress-induced tRNA degradation. Vacuolar Rny1 is segregated from cytoplasmic tRNAs in the absence of stress [7]. The exposure of yeast cells to oxidative stress results in the translocation of Rny1 and other vacuolar proteins into the cytoplasm, allowing them to access cytoplasmic tRNAs [7]. ANG is primarily localized in the nucleus under growth conditions [13]. Nuclear ANG stimulates rRNA transcription and cytoplasmic ANG is inhibited by ribonuclease/angiogenin inhibitor 1 (RNH1) [13]. In HeLa cells that are stressed with sodium arsenite, ANG translocates from the nucleus to the cytoplasm and accumulates in the SGs that are not associated with RNH1 [13]. Thus, after its change in localization, ANG can contribute to tiRNA production. In an earlier study, we reported the specific degradation of initiator tRNAMet (iMet) by the exonucleases Xrn1 and Xrn2 in HeLa cells under heat stress [14]. Because rapamycin inhibited iMet degradation under heat stress, we speculated that the mammalian target of rapamycin (mTOR), a rapamycin-binding protein, regulated the degradation of iMet in heat-stressed HeLa cells [14]. We wondered which factors contribute to the activation of Xrn2 under conditions of heat stress. In this study, we demonstrated that heat stress induced the diffusion of Xrn2 from the nucleolus to the nucleoplasm, suggesting that heat stress increased the association between Xrn2 and iMet. Importantly, mTOR knockdown
http://dx.doi.org/10.1016/j.febslet.2014.08.003 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
K. Watanabe et al. / FEBS Letters 588 (2014) 3454–3460
resulted in the suppression of Xrn2 diffusion under heat stress conditions. Our results indicate a translational-suppression mechanism involving mTOR-regulated iMet degradation in mammalian cells under heat stress.
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were transfected with 10 nM siRNA by using Lipofectamine RNAiMAX (Invitrogen) for 24 h at 37 °C in an atmosphere of 5% CO2. The following day, the cells were replated and then cultured for 24 h. 2.4. Northern blot analysis
2. Materials and methods 2.1. Cell culture conditions HeLa cells were maintained in RPMI1640 medium containing 10% fetal bovine serum (FBS; BioWest, France) and antibiotic-antimycotic (GIBCO) at 37 °C in an atmosphere of 5% CO2. Heat stress was inflicted by incubating the cells in a 43 °C water bath for 0–6 h. 2.2. Immunocytochemistry Immunocytochemistry was performed as described previously [14]. Mouse monoclonal anti-nucleolin (dilution 1:1000) and rabbit polyclonal anti-Xrn2 antibodies (dilution 1:2000) were purchased from Abcam. Alexa 594- and Alexa 647-conjugated secondary antibodies were purchased from Invitrogen, and 40 , 6-diamidino-2-phenylindole (DAPI) was obtained from Dojindo. The stained cells were examined using a confocal microscope (FV-1000; Olympus). 2.3. siRNA treatment To knock down mTOR, we used mTOR siRNA1 (sense sequence: GAG AAG AAA UGG AAG AAA UUU; antisense sequence: AUU UCU UCC AUU UCU UCU CUU) and mTOR siRNA2 (sense sequence: GUG CUG AAA UGC AGG AAU AUU, antisense: UAU UCC UGC AUU UCA GCA CUU) purchased from Thermo Fisher Scientific. HeLa cells
HeLa cells transfected with non-targeting siRNA (siControl) or the mTOR-specific siRNAs (siTOR) were treated with an RNA polymerase III inhibitor (50 lM; Merck) at 43 °C for 0–6 h. The cells were suspended in RNAiso (TaKaRa) for total RNA extraction according to the manufacturer’s instructions. Northern blot analysis was performed as previously described [14]. RNA was detected using a 50 -end [c-32P]-ATP-labeled oligonucleotide probe, and the blot was scanned using a FLA9000 imaging analyzer (Fujifilm). The sequences of the probes used in this study were as follows: 50 -GCA GAG GAT GGT TTC GAT CCA TC-30 for the detection of iMet and 50 -CAG GGT GGT ATG GCC GTA GAC-30 for the detection of 5S rRNA. Band intensities were quantified using Multi Gauge software (Fujifilm). Three independent replicates were performed and the results were averaged. 2.5. Western blot analysis After HeLa cells were transfected with specific siRNAs (siControl or siTOR) for 24 h, the cells were provided with fresh medium containing 10% FBS and then cultured for 24 h. HeLa cells transfected with specific siRNAs were then incubated at 43 °C for 0–6 h. The cells were suspended in cell lysis buffer [50 mM Tris–HCl pH 7.6, 150 mM KCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail (Merck)]. Protein concentrations were measured using the Bio-Rad Protein Assay kit (Bio-Rad). Cell lysates containing 10 lg of protein were
Fig. 1. Xrn2 diffuses into the nucleoplasm in response to heat stress. (A) The localization of the 50 –30 exoribonuclease Xrn2 in HeLa cells exposed to heat stress for the indicated times was monitored using immunofluorescent staining. Nuclear DNA was stained with 40 ,6-diamidino-2-phenylindole (DAPI). (B) The proportion of total cells that had Xrn2 strongly localized in the nucleolus (pink), distributed within the nucleus (yellow), or not localized to the nucleolus (blue). Two independent replicates were performed and the results were averaged (number of cells analyzed > 200). Scale bar = 10 lm.
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K. Watanabe et al. / FEBS Letters 588 (2014) 3454–3460
subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore). Blots were probed using the following primary antibodies: anti-mTOR (dilution 1:1000, Cell Signaling), anti-Xrn2 (dilution 1:2000, Abcam), or a-tubulin (dilution 1:1000, Cell Signaling). The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Millipore), and the target proteins were detected using a LAS4000mini imaging analyzer (Fujifilm) or C-DiGit Western blot scanner (LI-COR). Band intensities were quantified using the Multi Gauge program (Fujifilm) or image studio (LI-COR). 3. Results 3.1. Heat stress-induced translational suppression To determine whether overall translation was suppressed at 43 °C, the cells were pulse-labeled with L-[35S]methionine. 35 L-[ S]methionine incorporation was decreased by 30% under conditions of heat stress (Supplementary Fig. 1). This result shows that heat stress induced translational suppression.
we showed that Xrn2 is normally localized primarily in the nuclear regions not stained by DAPI, which likely represent nucleoli [14]. Upon heat stress, Xrn2 disperses into the nucleoplasm [14]. In this study, we performed immunocytochemistry to determine whether Xrn2 and the nucleolar protein nucleolin are colocalized under conditions of heat stress. Although nucleolin diffuses from the nucleolus to the nucleoplasm under heat stress [17], it localizes in the nucleolus in non-stressed cells. Wang et al. reported that nucleolin rapidly diffuses within the nucleoplasm after 5–30 min of heat stress exposure and relocates to the nucleolus after 90 min [18]. Although nucleolin diffused into the nucleoplasm after 3 h of heat stress exposure, it was strongly and specifically localized at the periphery of nuclei under conditions of heat stress (Fig. 1A). Xrn2 was primarily localized to the nucleolus, where it colocalized with nucleolin under normal conditions (Fig. 1A and B). Exposure of the cells to heat stress resulted in rapid diffusion of Xrn2 into the nucleoplasm; eventually, it was no longer localized in the nucleolus (Fig. 1A and B). In our previous study [14,19], we showed that iMet was distributed throughout the nucleus including the nucleolus. Therefore, we speculate that iMet interacts with Xrn2 that diffuses into the nucleoplasm under conditions of heat stress.
3.2. Nucleolus-localized Xrn2 diffuses into the nucleoplasm upon heat stress
3.3. mTOR regulates iMet degradation under heat stress conditions
Xrn2 is localized within the nucleus including the nucleolus under normal culture conditions [15,16]. In our previous study,
In our previous study, we reported that rapamycin inhibited the degradation of iMet under conditions of heat stress [14].
Fig. 2. Degradation of iMet in mTOR-silenced cells under heat stress. (A) The efficiency of knockdown by non-targeting (siControl) or mTOR-specific (siTOR) siRNAs was determined using Western blot analysis. The relative mTOR expression levels were normalized to the levels of a-tubulin. Two independent replicates were performed, and the results were averaged. (B) Northern blot analysis of initiator tRNAMet (iMet) degradation in cells treated with siControl or siTOR under heat stress. Total RNA (7.5 lg) was loaded and blotted. iMet and 5S rRNA were detected using specific probes. (C) Degradation of iMet was measured in cells treated with siControl and siTOR. Circle, siControltreated; square, siTOR-treated. The degradation of iMet was normalized to 5S rRNA levels. Data represent the mean ± standard error of the mean (S.E.M.) of three independent experiments.
K. Watanabe et al. / FEBS Letters 588 (2014) 3454–3460
Rapamycin inhibits the phosphorylation activity of mTOR [20]. Therefore, we assessed whether knockdown of mTOR suppresses or enhances the degradation of iMet. mTOR protein levels were measured in HeLa cells treated with siTOR by Western blotting (Fig. 2A). Two independent replicates were performed and averaged. siTOR decreased the expression of mTOR to 6% of that observed in the siControl-treated cells. Next, we performed a northern blot analysis to assess the degradation of iMet in mTOR-silenced cells exposed to heat stress. As shown in Fig. 2B and C, the degradation of iMet was suppressed in mTOR-silenced cells under conditions of heat stress. The degradation pattern of iMet in mTOR-silenced cells was similar to that observed in Xrn2-silenced cells, as reported previously [14]. In our previous study, we showed that Xrn2 is the primary nuclease responsible for the degradation of iMet under conditions of heat stress [14]. These results indicate that the ribonucleolytic activity of Xrn2 is suppressed by mTOR knockdown, suggesting that mTOR contributes to the regulation of Xrn2 activity. 3.4. Xrn2 protein expression is affected by mTOR under conditions of heat stress The mechanism underlying the mTOR knockdown-induced suppression of iMet degradation is unclear. We speculated that mTOR knockdown may induce a decrease in Xrn2 protein expression levels during heat stress. Therefore, we used Western blot analysis to assess Xrn2 protein levels in mTOR-silenced cells under heat stress.
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Prior to application of heat, Xrn2 protein levels were not affected by mTOR knockdown (Fig. 3A), suggesting that Xrn2 protein expression is not regulated by mTOR under normal conditions. After the temperature shift, Xrn2 protein levels in cells treated with siControl remained unaltered (Fig. 3B and C). In contrast, heat stress decreased Xrn2 protein levels in cells treated with siTOR (Fig. 3B and C). The suppression of iMet degradation seems to be one of the reasons for the Xrn2 protein decrease in mTOR-silenced cells under heat stress. 3.5. mTOR controls the diffusion of Xrn2 into the nucleoplasm under conditions of heat stress Heat stress induced diffusion of Xrn2 to the nucleoplasm (Fig. 1), which appeared to facilitate the iMet–Xrn2 interaction and iMet degradation. We hypothesized that the inhibition of heat stress-induced iMet degradation in mTOR-silenced cells (Fig. 2B and C) resulted from inhibition of the diffusion of Xrn2 to the nucleoplasm. To determine whether the diffusion of Xrn2 into the nucleoplasm is inhibited by siTOR treatment, we performed immunocytochemistry (Fig. 4). The diffusion pattern of Xrn2 and nucleolin in siControl-treated cells was similar to that observed in the absence of siRNA treatment (Figs. 1 and 4A). In contrast, Xrn2 was more predominantly localized to the nucleolus in siTOR-treated cells under heat stress compared with siControltreated cells (Fig. 4A and B). In addition, nucleolin was localized in the nucleolus of siTOR-treated cells under heat stress (Fig. 4B).
Fig. 3. Levels of Xrn2 protein in mTOR-silenced cells. (A) After HeLa cells were transfected for 24 h, fresh medium containing 10% FBS was added, and the cells were cultured for a further 48 h. Xrn2 protein levels were analyzed by Western blot. Xrn2 protein levels in cells treated with siControl or siTOR were compared. a-Tubulin was used as a loading control. Data represent the mean ± S.E.M. of three independent experiments. (B) Xrn2 protein levels in heat-stressed cells treated with siControl or siTOR were analyzed by Western blot analysis. a-Tubulin was used as a control. (C) Relative Xrn2 protein levels in cells treated with siControl (filled circle) or siTOR (filled square). The expression of Xrn2 was normalized to that of a-tubulin. Data represent the mean ± S.E.M. of five independent experiments. ⁄⁄P < 0.01; P-values were calculated using Student’s t-test by comparing siControl with siTOR.
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Furthermore, the nucleoplasmic diffusion of Xrn2 was inhibited in the presence of rapamycin under heat stress (Supplementary Fig. 2). These results suggest that the altered localization of Xrn2 under conditions of heat stress was regulated by mTOR. The results shown in Figs. 2B, C, and 4 suggest that iMet degradation was suppressed under conditions of heat stress in mTOR-silenced cells because the interaction with Xrn2 was disrupted. Therefore, we conclude that mTOR knockdown induced a decrease in Xrn2
protein levels and induced nucleoplasmic diffusion of Xrn2 under conditions of heat stress, thereby suppressing the degradation of iMet. 4. Discussion Here, we showed that heat stress induced the diffusion of Xrn2 from the nucleolus to the nucleoplasm, leading to the interaction
Fig. 4. Immunofluorescence of Xrn2 in mTOR-silenced cells under heat stress. HeLa cells transfected with siControl or siTOR were exposed to heat stress. The localization of Xrn2 in cells treated with siControl (A) or siTOR (B) was analyzed by immunocytochemistry. Proportion of total cells that had Xrn2 strongly localized in the nucleolus (pink), distributed within the nucleus (yellow), or not localized in the nucleolus (blue). Two independent replicates were performed, and the results were averaged (number of cells analyzed > 200). Scale bar = 10 lm.
K. Watanabe et al. / FEBS Letters 588 (2014) 3454–3460
between Xrn2 and iMet. Our results indicate that mTOR regulates the diffusion of Xrn2 into the nucleoplasm under conditions of heat stress. We showed that heat stress induced the diffusion of Xrn2, facilitating the degradation of iMet (Figs. 1, 2B and 4). Oxidative stress reportedly alters the localization of RNases; Rny1 in yeast diffuses from the vacuole to the cytoplasm [7] and ANG in mammalian cells translocates from the nucleus to SGs [13]. Thus, Rny1 and ANG interact with tRNAs by altering their localization, leading to their degradation. Even if the synthesis of these nucleases is increased after the exposure of cells to stress, the tRNAs are not rapidly degraded by the nucleases, which have different localizations. Therefore, the simple system of activating these nucleases by altering their localization was probably adopted as a stress response. Heat stress induced the diffusion of Xrn2 into the nucleoplasm, facilitating the degradation of iMet (Figs. 1, 2B and 4). However, the mechanism of Xrn2 activation may not be explained by the diffusion of Xrn2 into the nucleoplasm alone, because iMet is distributed throughout the nucleus including the nucleolus [14,15]. The NetworKIN database (http://networkin.info/version_2_0/search. php) predicts that multiple sites in Xrn2 are phosphorylated by a stress-activated protein kinase. Xrn2 reportedly interacts with eIF2AK, which phosphorylates the translation initiation factor eIF2S1 [21]. In addition, the STRING database (http://string-db. org/) shows that eIF2AK is related to mTOR through several protein–protein interactions. The content of these databases and work conducted by Dorf and colleagues [21] suggest that heat stress induces the modification of amino acid(s) in Xrn2 through enzyme(s) such as eIF2AK and stress-activated protein kinase, and modified Xrn2 is activated.mTOR interacts with different partner proteins to form at least two distinct signaling complexes, the mTOR complex 1 (mTORC1) and mTORC2 [22]. Acute rapamycin treatment inhibits the catalytic activity and signaling capacity of mTORC1 but not mTORC2 [22,23]. Our current studies indicate that the degradation of iMet is regulated by mTORC1 because rapamycin inhibited the degradation of iMet and the nucleoplasmic diffusion of Xrn2 under heat stress (Fig. 2B, C and Supplementary Fig. 2). Currently, the major stress-induced translational suppression mechanisms are categorized as follows: (I) phosphorylation of eIF2a, (II) formation of SGs, (III) translational suppression by tiRNAs, and (IV) signaling through the mTOR pathway. Because eIF2a is phosphorylated under stress, it fails to form a specific complex with iMet, thereby inhibiting the translational initiation step of protein biosynthesis [3]. Various stressors induce the formation of SGs including the exonuclease Xrn1 that degrades mRNA and promotes the accumulation of mRNAs into SGs [1,24,25]. Angiogenin induces 50 -tiRNAs to inhibit translation by interfering with several functions of eIF4G [10]. The mTOR signaling pathway, including the eIF4E binding/inhibiting protein (4E-BP) and ribosomal protein S6 kinase 1 (S6K1), regulates the translation initiation step in protein synthesis under stress conditions [26,27]. Under these conditions, the translation enhancer 4E-BP is phosphorylated by mTOR, inhibiting the assembly of the eIF4E/eIF4F complex and blocking translation [28,29]. mTOR-phosphorylated S6K1 in turn phosphorylates the ribosomal protein S6, the initiation factor eIF4B, and the translation elongation factor eEF2 kinase, inhibiting protein translation [30,31]. Our results suggest a translational-regulation mechanism through mTOR-regulated iMet degradation under conditions of heat stress. Our findings, together with recent reports, indicate that the stress-induced translational regulation mediated by tRNA degradation differs in the context of heat and oxidative stress in mammalian cells. Thus, our current study contributes to the understanding of the stress-response mechanism.
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Acknowledgment This work was supported in part by the Naito Foundation to K.W. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014.08. 003. References [1] Kedersha, N., Chen, S., Gilks, N., Li, W., Miller, I.J., Stahl, J. and Anderson, P. (2002) Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol. Biol. Cell 13, 195–210. [2] Velichko, A.K., Markova, E.N., Petrova, N.V., Razin, S.V. and Kantidze, O.L. (2013) Mechanisms of heat shock response in mammals. Cell. Mol. Life Sci., http://dx.doi.org/10.1007/s00018-013-1348-7. [3] Hofmann, S., Cherkasova, V., Bankhead, P., Bukaku, B. and Stoecklin, G. (2012) Translation suppression promotes stress granule formation and cell survival in response to cold shock. Mol. Biol. Cell 48 (1), 3786–3800. [4] Gardner, L.B. (2010) Nonsense-mediated RNA decay regulation by cellular stress: implications for tumorigenesis. Mol. Cancer Res. 8, 295–308. [5] Netzer, N., Goodenbour, J.M., David, A., Dittmar, K.A., Jones, R.B., Schneider, J.R., Boone, D., Eves, E.M., Rosner, M.R., Gibbs, J.S., Embry, A., Dolan, B., Das, S., Hickman, H.D., Berglund, P., Bennink, J.R., Yewdell, J.W. and Tao Pan, T. (2009) Innate immune and chemically triggered oxidative stress modifies translational fidelity. Nature 462, 522–526. [6] Thompson, D.M., Lu, C., Green, P.J. and Parker, R. (2008) TRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA 14, 2095–2103. [7] Thompson, D.M. and Parker, R. (2009) The RNase Rny1p cleaves tRNAs and promotes cell death during oxidative stress in Saccharomyces cerevisiae. J. Cell Biol. 185, 43–50. [8] Fu, H., Feng, J., Liu, Q., Sun, F., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X. (2009) Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Lett. 583, 437–442. [9] Yamasaki, S., Ivanov, P., Hu, G.F. and Anderson, P. (2009) Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35–42. [10] Ivanov, P., Emara, M.M., Villen, J., Gygi, S.P. and Anderson, P. (2011) Angiogenin-induced tRNA fragments inhibit translation initiation. Mol. Cell 43, 613–623. [11] Emara, M.M., Ivanov, P., Hickman, T., Dawra, N., Tisdale, S., Kedersha, N., Hu, G. and Anderson, P. (2010) Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J. Biol. Chem. 285, 10959–10968. [12] Yamasaki, S. and Anderson, P. (2008) Reprogramming mRNA translation during stress. Curr. Opin. Cell Biol. 185, 222–226. [13] Pizzo, E., Sarcinelli, C., Sheng, J., Fusco, S., Formiggini, F., Netti, P., Yu, W., D’Alessio, G. and Hu, G.F. (2013) Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin and controls its growth and survival activities. J. Cell Sci. 126 (18), 4308–4319. [14] Watanabe, K., Miyagawa, R., Tomikawa, C., Mizuno, R., Takahashi, A., Hori, H. and Ijiri, K. (2013) Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells. Nucleic Acids Res. 41 (8), 4671–4685. [15] Scherl, A., Couté, Y., Déon, C., Callé, A., Kindbeiter, K., Sachez, J.C., Greco, A., Hochstrasser, D. and Diaz, J.J. (2002) Functional proteomic analysis of human nucleolus. Mol. Biol. Cell 13 (11), 4100–4109. [16] Wang, M. and Pestov, D.G. (2011) 50 -end surveillance by Xrn2 acts as a shared mechanism for mammalian pre-rRNA maturation and decay. Nucleic Acids Res. 39 (5), 1811–1822. [17] Daniely, Y. and Borowiec, J.A. (2000) Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication. J. Cell Biol. 149 (4), 799–810. [18] Wang, Y., Guan, J., Wang, H., Wang, Y., Leeper, D. and Iliakis, G. (2001) Regulation of DNA replication after heat shock by replication protein Anucleolin interactions. J. Biol. Chem. 276 (23), 20579–20588. [19] Miyagawa, R., Mizuno, R., Watanabe, K. and Ijiri, K. (2012) Formation of tRNA granules in the nucleus of heat-induced human cells. Biochem. Biophys. Res. Commun. 418, 149–155. [20] Hartford, C.M. and Ratain, M.J. (2007) Rapamycin: something old, something new, sometimes borrowed and now renewed. Clin. Pharmacol. Ther. 82 (4), 381–388. [21] Li, S., Wang, L., Berman, M., Kong, Y.Y. and Dorf, M.E. (2011) Mapping a dynamic innate immunity protein interaction network regulating type I interferon production. Immunity 35 (3), 426–440.
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mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress Kazunori Watanabe a,b,⇑, Kenichi Ijiri b, Takashi Ohtsuki a a b
Department of Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan Radioisotope Center, The University of Tokyo, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 18 June 2014 Revised 4 August 2014 Accepted 4 August 2014 Available online 12 August 2014 Edited by Michael Ibba Keywords: Mammalian target of rapamycin tRNA degradation Xrn2 Heat stress
a b s t r a c t Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNAMet (iMet) through 50 –30 exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Eukaryotic cells possess mechanisms that respond to environmental stresses, including heat, oxidation, and nutrient starvation. The major stress-response mechanisms include DNA damage repair, cell-cycle arrest, and translational suppression [1,2]. The upregulation of heat shock factors, modification of specific proteins, formation of stress granules (SGs) and nuclear stress bodies, and modulation of RNA metabolism and translocation are important components of the stress response [1–4]. Transfer RNA (tRNA) is a fundamental component of the translation machinery that plays a key role in modulating translation during cellular stress. For instance, non-methionine-tRNAs (e.g., tRNALeu) are aminoacylated with methionine in HeLa cells under conditions of oxidative stress. The subsequent increase in mischarged tRNAs leads to the misincorporation of methionine during protein synthesis [5]. Moreover, tRNA fragments are produced by the endonuclease Rny1 in response to nutrient starvation and oxidative stress in yeast [6,7]. In mammalian cells under oxidative stress, tRNA fragments (tiRNAs) generated by angiogenin (ANG) endonuclease suppress global protein translation [8–10]. In addition, tiRNAs trigger the assembly of stress granules, which transiently store untranslated mRNA–protein complexes in U2OS ⇑ Corresponding author at: Department of Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan. E-mail address: [email protected] (K. Watanabe).
cells [11]. The formation of SGs is an important mechanism by which cells reprogram translation to survive in adverse conditions [12]. The activation of Rny1 in yeast and ANG in mammalian cells is important for oxidative stress-induced tRNA degradation. Vacuolar Rny1 is segregated from cytoplasmic tRNAs in the absence of stress [7]. The exposure of yeast cells to oxidative stress results in the translocation of Rny1 and other vacuolar proteins into the cytoplasm, allowing them to access cytoplasmic tRNAs [7]. ANG is primarily localized in the nucleus under growth conditions [13]. Nuclear ANG stimulates rRNA transcription and cytoplasmic ANG is inhibited by ribonuclease/angiogenin inhibitor 1 (RNH1) [13]. In HeLa cells that are stressed with sodium arsenite, ANG translocates from the nucleus to the cytoplasm and accumulates in the SGs that are not associated with RNH1 [13]. Thus, after its change in localization, ANG can contribute to tiRNA production. In an earlier study, we reported the specific degradation of initiator tRNAMet (iMet) by the exonucleases Xrn1 and Xrn2 in HeLa cells under heat stress [14]. Because rapamycin inhibited iMet degradation under heat stress, we speculated that the mammalian target of rapamycin (mTOR), a rapamycin-binding protein, regulated the degradation of iMet in heat-stressed HeLa cells [14]. We wondered which factors contribute to the activation of Xrn2 under conditions of heat stress. In this study, we demonstrated that heat stress induced the diffusion of Xrn2 from the nucleolus to the nucleoplasm, suggesting that heat stress increased the association between Xrn2 and iMet. Importantly, mTOR knockdown
http://dx.doi.org/10.1016/j.febslet.2014.08.003 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
K. Watanabe et al. / FEBS Letters 588 (2014) 3454–3460
resulted in the suppression of Xrn2 diffusion under heat stress conditions. Our results indicate a translational-suppression mechanism involving mTOR-regulated iMet degradation in mammalian cells under heat stress.
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were transfected with 10 nM siRNA by using Lipofectamine RNAiMAX (Invitrogen) for 24 h at 37 °C in an atmosphere of 5% CO2. The following day, the cells were replated and then cultured for 24 h. 2.4. Northern blot analysis
2. Materials and methods 2.1. Cell culture conditions HeLa cells were maintained in RPMI1640 medium containing 10% fetal bovine serum (FBS; BioWest, France) and antibiotic-antimycotic (GIBCO) at 37 °C in an atmosphere of 5% CO2. Heat stress was inflicted by incubating the cells in a 43 °C water bath for 0–6 h. 2.2. Immunocytochemistry Immunocytochemistry was performed as described previously [14]. Mouse monoclonal anti-nucleolin (dilution 1:1000) and rabbit polyclonal anti-Xrn2 antibodies (dilution 1:2000) were purchased from Abcam. Alexa 594- and Alexa 647-conjugated secondary antibodies were purchased from Invitrogen, and 40 , 6-diamidino-2-phenylindole (DAPI) was obtained from Dojindo. The stained cells were examined using a confocal microscope (FV-1000; Olympus). 2.3. siRNA treatment To knock down mTOR, we used mTOR siRNA1 (sense sequence: GAG AAG AAA UGG AAG AAA UUU; antisense sequence: AUU UCU UCC AUU UCU UCU CUU) and mTOR siRNA2 (sense sequence: GUG CUG AAA UGC AGG AAU AUU, antisense: UAU UCC UGC AUU UCA GCA CUU) purchased from Thermo Fisher Scientific. HeLa cells
HeLa cells transfected with non-targeting siRNA (siControl) or the mTOR-specific siRNAs (siTOR) were treated with an RNA polymerase III inhibitor (50 lM; Merck) at 43 °C for 0–6 h. The cells were suspended in RNAiso (TaKaRa) for total RNA extraction according to the manufacturer’s instructions. Northern blot analysis was performed as previously described [14]. RNA was detected using a 50 -end [c-32P]-ATP-labeled oligonucleotide probe, and the blot was scanned using a FLA9000 imaging analyzer (Fujifilm). The sequences of the probes used in this study were as follows: 50 -GCA GAG GAT GGT TTC GAT CCA TC-30 for the detection of iMet and 50 -CAG GGT GGT ATG GCC GTA GAC-30 for the detection of 5S rRNA. Band intensities were quantified using Multi Gauge software (Fujifilm). Three independent replicates were performed and the results were averaged. 2.5. Western blot analysis After HeLa cells were transfected with specific siRNAs (siControl or siTOR) for 24 h, the cells were provided with fresh medium containing 10% FBS and then cultured for 24 h. HeLa cells transfected with specific siRNAs were then incubated at 43 °C for 0–6 h. The cells were suspended in cell lysis buffer [50 mM Tris–HCl pH 7.6, 150 mM KCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail (Merck)]. Protein concentrations were measured using the Bio-Rad Protein Assay kit (Bio-Rad). Cell lysates containing 10 lg of protein were
Fig. 1. Xrn2 diffuses into the nucleoplasm in response to heat stress. (A) The localization of the 50 –30 exoribonuclease Xrn2 in HeLa cells exposed to heat stress for the indicated times was monitored using immunofluorescent staining. Nuclear DNA was stained with 40 ,6-diamidino-2-phenylindole (DAPI). (B) The proportion of total cells that had Xrn2 strongly localized in the nucleolus (pink), distributed within the nucleus (yellow), or not localized to the nucleolus (blue). Two independent replicates were performed and the results were averaged (number of cells analyzed > 200). Scale bar = 10 lm.
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subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore). Blots were probed using the following primary antibodies: anti-mTOR (dilution 1:1000, Cell Signaling), anti-Xrn2 (dilution 1:2000, Abcam), or a-tubulin (dilution 1:1000, Cell Signaling). The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Millipore), and the target proteins were detected using a LAS4000mini imaging analyzer (Fujifilm) or C-DiGit Western blot scanner (LI-COR). Band intensities were quantified using the Multi Gauge program (Fujifilm) or image studio (LI-COR). 3. Results 3.1. Heat stress-induced translational suppression To determine whether overall translation was suppressed at 43 °C, the cells were pulse-labeled with L-[35S]methionine. 35 L-[ S]methionine incorporation was decreased by 30% under conditions of heat stress (Supplementary Fig. 1). This result shows that heat stress induced translational suppression.
we showed that Xrn2 is normally localized primarily in the nuclear regions not stained by DAPI, which likely represent nucleoli [14]. Upon heat stress, Xrn2 disperses into the nucleoplasm [14]. In this study, we performed immunocytochemistry to determine whether Xrn2 and the nucleolar protein nucleolin are colocalized under conditions of heat stress. Although nucleolin diffuses from the nucleolus to the nucleoplasm under heat stress [17], it localizes in the nucleolus in non-stressed cells. Wang et al. reported that nucleolin rapidly diffuses within the nucleoplasm after 5–30 min of heat stress exposure and relocates to the nucleolus after 90 min [18]. Although nucleolin diffused into the nucleoplasm after 3 h of heat stress exposure, it was strongly and specifically localized at the periphery of nuclei under conditions of heat stress (Fig. 1A). Xrn2 was primarily localized to the nucleolus, where it colocalized with nucleolin under normal conditions (Fig. 1A and B). Exposure of the cells to heat stress resulted in rapid diffusion of Xrn2 into the nucleoplasm; eventually, it was no longer localized in the nucleolus (Fig. 1A and B). In our previous study [14,19], we showed that iMet was distributed throughout the nucleus including the nucleolus. Therefore, we speculate that iMet interacts with Xrn2 that diffuses into the nucleoplasm under conditions of heat stress.
3.2. Nucleolus-localized Xrn2 diffuses into the nucleoplasm upon heat stress
3.3. mTOR regulates iMet degradation under heat stress conditions
Xrn2 is localized within the nucleus including the nucleolus under normal culture conditions [15,16]. In our previous study,
In our previous study, we reported that rapamycin inhibited the degradation of iMet under conditions of heat stress [14].
Fig. 2. Degradation of iMet in mTOR-silenced cells under heat stress. (A) The efficiency of knockdown by non-targeting (siControl) or mTOR-specific (siTOR) siRNAs was determined using Western blot analysis. The relative mTOR expression levels were normalized to the levels of a-tubulin. Two independent replicates were performed, and the results were averaged. (B) Northern blot analysis of initiator tRNAMet (iMet) degradation in cells treated with siControl or siTOR under heat stress. Total RNA (7.5 lg) was loaded and blotted. iMet and 5S rRNA were detected using specific probes. (C) Degradation of iMet was measured in cells treated with siControl and siTOR. Circle, siControltreated; square, siTOR-treated. The degradation of iMet was normalized to 5S rRNA levels. Data represent the mean ± standard error of the mean (S.E.M.) of three independent experiments.
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Rapamycin inhibits the phosphorylation activity of mTOR [20]. Therefore, we assessed whether knockdown of mTOR suppresses or enhances the degradation of iMet. mTOR protein levels were measured in HeLa cells treated with siTOR by Western blotting (Fig. 2A). Two independent replicates were performed and averaged. siTOR decreased the expression of mTOR to 6% of that observed in the siControl-treated cells. Next, we performed a northern blot analysis to assess the degradation of iMet in mTOR-silenced cells exposed to heat stress. As shown in Fig. 2B and C, the degradation of iMet was suppressed in mTOR-silenced cells under conditions of heat stress. The degradation pattern of iMet in mTOR-silenced cells was similar to that observed in Xrn2-silenced cells, as reported previously [14]. In our previous study, we showed that Xrn2 is the primary nuclease responsible for the degradation of iMet under conditions of heat stress [14]. These results indicate that the ribonucleolytic activity of Xrn2 is suppressed by mTOR knockdown, suggesting that mTOR contributes to the regulation of Xrn2 activity. 3.4. Xrn2 protein expression is affected by mTOR under conditions of heat stress The mechanism underlying the mTOR knockdown-induced suppression of iMet degradation is unclear. We speculated that mTOR knockdown may induce a decrease in Xrn2 protein expression levels during heat stress. Therefore, we used Western blot analysis to assess Xrn2 protein levels in mTOR-silenced cells under heat stress.
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Prior to application of heat, Xrn2 protein levels were not affected by mTOR knockdown (Fig. 3A), suggesting that Xrn2 protein expression is not regulated by mTOR under normal conditions. After the temperature shift, Xrn2 protein levels in cells treated with siControl remained unaltered (Fig. 3B and C). In contrast, heat stress decreased Xrn2 protein levels in cells treated with siTOR (Fig. 3B and C). The suppression of iMet degradation seems to be one of the reasons for the Xrn2 protein decrease in mTOR-silenced cells under heat stress. 3.5. mTOR controls the diffusion of Xrn2 into the nucleoplasm under conditions of heat stress Heat stress induced diffusion of Xrn2 to the nucleoplasm (Fig. 1), which appeared to facilitate the iMet–Xrn2 interaction and iMet degradation. We hypothesized that the inhibition of heat stress-induced iMet degradation in mTOR-silenced cells (Fig. 2B and C) resulted from inhibition of the diffusion of Xrn2 to the nucleoplasm. To determine whether the diffusion of Xrn2 into the nucleoplasm is inhibited by siTOR treatment, we performed immunocytochemistry (Fig. 4). The diffusion pattern of Xrn2 and nucleolin in siControl-treated cells was similar to that observed in the absence of siRNA treatment (Figs. 1 and 4A). In contrast, Xrn2 was more predominantly localized to the nucleolus in siTOR-treated cells under heat stress compared with siControltreated cells (Fig. 4A and B). In addition, nucleolin was localized in the nucleolus of siTOR-treated cells under heat stress (Fig. 4B).
Fig. 3. Levels of Xrn2 protein in mTOR-silenced cells. (A) After HeLa cells were transfected for 24 h, fresh medium containing 10% FBS was added, and the cells were cultured for a further 48 h. Xrn2 protein levels were analyzed by Western blot. Xrn2 protein levels in cells treated with siControl or siTOR were compared. a-Tubulin was used as a loading control. Data represent the mean ± S.E.M. of three independent experiments. (B) Xrn2 protein levels in heat-stressed cells treated with siControl or siTOR were analyzed by Western blot analysis. a-Tubulin was used as a control. (C) Relative Xrn2 protein levels in cells treated with siControl (filled circle) or siTOR (filled square). The expression of Xrn2 was normalized to that of a-tubulin. Data represent the mean ± S.E.M. of five independent experiments. ⁄⁄P < 0.01; P-values were calculated using Student’s t-test by comparing siControl with siTOR.
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Furthermore, the nucleoplasmic diffusion of Xrn2 was inhibited in the presence of rapamycin under heat stress (Supplementary Fig. 2). These results suggest that the altered localization of Xrn2 under conditions of heat stress was regulated by mTOR. The results shown in Figs. 2B, C, and 4 suggest that iMet degradation was suppressed under conditions of heat stress in mTOR-silenced cells because the interaction with Xrn2 was disrupted. Therefore, we conclude that mTOR knockdown induced a decrease in Xrn2
protein levels and induced nucleoplasmic diffusion of Xrn2 under conditions of heat stress, thereby suppressing the degradation of iMet. 4. Discussion Here, we showed that heat stress induced the diffusion of Xrn2 from the nucleolus to the nucleoplasm, leading to the interaction
Fig. 4. Immunofluorescence of Xrn2 in mTOR-silenced cells under heat stress. HeLa cells transfected with siControl or siTOR were exposed to heat stress. The localization of Xrn2 in cells treated with siControl (A) or siTOR (B) was analyzed by immunocytochemistry. Proportion of total cells that had Xrn2 strongly localized in the nucleolus (pink), distributed within the nucleus (yellow), or not localized in the nucleolus (blue). Two independent replicates were performed, and the results were averaged (number of cells analyzed > 200). Scale bar = 10 lm.
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between Xrn2 and iMet. Our results indicate that mTOR regulates the diffusion of Xrn2 into the nucleoplasm under conditions of heat stress. We showed that heat stress induced the diffusion of Xrn2, facilitating the degradation of iMet (Figs. 1, 2B and 4). Oxidative stress reportedly alters the localization of RNases; Rny1 in yeast diffuses from the vacuole to the cytoplasm [7] and ANG in mammalian cells translocates from the nucleus to SGs [13]. Thus, Rny1 and ANG interact with tRNAs by altering their localization, leading to their degradation. Even if the synthesis of these nucleases is increased after the exposure of cells to stress, the tRNAs are not rapidly degraded by the nucleases, which have different localizations. Therefore, the simple system of activating these nucleases by altering their localization was probably adopted as a stress response. Heat stress induced the diffusion of Xrn2 into the nucleoplasm, facilitating the degradation of iMet (Figs. 1, 2B and 4). However, the mechanism of Xrn2 activation may not be explained by the diffusion of Xrn2 into the nucleoplasm alone, because iMet is distributed throughout the nucleus including the nucleolus [14,15]. The NetworKIN database (http://networkin.info/version_2_0/search. php) predicts that multiple sites in Xrn2 are phosphorylated by a stress-activated protein kinase. Xrn2 reportedly interacts with eIF2AK, which phosphorylates the translation initiation factor eIF2S1 [21]. In addition, the STRING database (http://string-db. org/) shows that eIF2AK is related to mTOR through several protein–protein interactions. The content of these databases and work conducted by Dorf and colleagues [21] suggest that heat stress induces the modification of amino acid(s) in Xrn2 through enzyme(s) such as eIF2AK and stress-activated protein kinase, and modified Xrn2 is activated.mTOR interacts with different partner proteins to form at least two distinct signaling complexes, the mTOR complex 1 (mTORC1) and mTORC2 [22]. Acute rapamycin treatment inhibits the catalytic activity and signaling capacity of mTORC1 but not mTORC2 [22,23]. Our current studies indicate that the degradation of iMet is regulated by mTORC1 because rapamycin inhibited the degradation of iMet and the nucleoplasmic diffusion of Xrn2 under heat stress (Fig. 2B, C and Supplementary Fig. 2). Currently, the major stress-induced translational suppression mechanisms are categorized as follows: (I) phosphorylation of eIF2a, (II) formation of SGs, (III) translational suppression by tiRNAs, and (IV) signaling through the mTOR pathway. Because eIF2a is phosphorylated under stress, it fails to form a specific complex with iMet, thereby inhibiting the translational initiation step of protein biosynthesis [3]. Various stressors induce the formation of SGs including the exonuclease Xrn1 that degrades mRNA and promotes the accumulation of mRNAs into SGs [1,24,25]. Angiogenin induces 50 -tiRNAs to inhibit translation by interfering with several functions of eIF4G [10]. The mTOR signaling pathway, including the eIF4E binding/inhibiting protein (4E-BP) and ribosomal protein S6 kinase 1 (S6K1), regulates the translation initiation step in protein synthesis under stress conditions [26,27]. Under these conditions, the translation enhancer 4E-BP is phosphorylated by mTOR, inhibiting the assembly of the eIF4E/eIF4F complex and blocking translation [28,29]. mTOR-phosphorylated S6K1 in turn phosphorylates the ribosomal protein S6, the initiation factor eIF4B, and the translation elongation factor eEF2 kinase, inhibiting protein translation [30,31]. Our results suggest a translational-regulation mechanism through mTOR-regulated iMet degradation under conditions of heat stress. Our findings, together with recent reports, indicate that the stress-induced translational regulation mediated by tRNA degradation differs in the context of heat and oxidative stress in mammalian cells. Thus, our current study contributes to the understanding of the stress-response mechanism.
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Acknowledgment This work was supported in part by the Naito Foundation to K.W. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014.08. 003. References [1] Kedersha, N., Chen, S., Gilks, N., Li, W., Miller, I.J., Stahl, J. and Anderson, P. (2002) Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol. Biol. Cell 13, 195–210. [2] Velichko, A.K., Markova, E.N., Petrova, N.V., Razin, S.V. and Kantidze, O.L. (2013) Mechanisms of heat shock response in mammals. Cell. Mol. Life Sci., http://dx.doi.org/10.1007/s00018-013-1348-7. [3] Hofmann, S., Cherkasova, V., Bankhead, P., Bukaku, B. and Stoecklin, G. (2012) Translation suppression promotes stress granule formation and cell survival in response to cold shock. Mol. Biol. Cell 48 (1), 3786–3800. [4] Gardner, L.B. (2010) Nonsense-mediated RNA decay regulation by cellular stress: implications for tumorigenesis. Mol. Cancer Res. 8, 295–308. [5] Netzer, N., Goodenbour, J.M., David, A., Dittmar, K.A., Jones, R.B., Schneider, J.R., Boone, D., Eves, E.M., Rosner, M.R., Gibbs, J.S., Embry, A., Dolan, B., Das, S., Hickman, H.D., Berglund, P., Bennink, J.R., Yewdell, J.W. and Tao Pan, T. (2009) Innate immune and chemically triggered oxidative stress modifies translational fidelity. Nature 462, 522–526. [6] Thompson, D.M., Lu, C., Green, P.J. and Parker, R. (2008) TRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA 14, 2095–2103. [7] Thompson, D.M. and Parker, R. (2009) The RNase Rny1p cleaves tRNAs and promotes cell death during oxidative stress in Saccharomyces cerevisiae. J. Cell Biol. 185, 43–50. [8] Fu, H., Feng, J., Liu, Q., Sun, F., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X. (2009) Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Lett. 583, 437–442. [9] Yamasaki, S., Ivanov, P., Hu, G.F. and Anderson, P. (2009) Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35–42. [10] Ivanov, P., Emara, M.M., Villen, J., Gygi, S.P. and Anderson, P. (2011) Angiogenin-induced tRNA fragments inhibit translation initiation. Mol. Cell 43, 613–623. [11] Emara, M.M., Ivanov, P., Hickman, T., Dawra, N., Tisdale, S., Kedersha, N., Hu, G. and Anderson, P. (2010) Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J. Biol. Chem. 285, 10959–10968. [12] Yamasaki, S. and Anderson, P. (2008) Reprogramming mRNA translation during stress. Curr. Opin. Cell Biol. 185, 222–226. [13] Pizzo, E., Sarcinelli, C., Sheng, J., Fusco, S., Formiggini, F., Netti, P., Yu, W., D’Alessio, G. and Hu, G.F. (2013) Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin and controls its growth and survival activities. J. Cell Sci. 126 (18), 4308–4319. [14] Watanabe, K., Miyagawa, R., Tomikawa, C., Mizuno, R., Takahashi, A., Hori, H. and Ijiri, K. (2013) Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells. Nucleic Acids Res. 41 (8), 4671–4685. [15] Scherl, A., Couté, Y., Déon, C., Callé, A., Kindbeiter, K., Sachez, J.C., Greco, A., Hochstrasser, D. and Diaz, J.J. (2002) Functional proteomic analysis of human nucleolus. Mol. Biol. Cell 13 (11), 4100–4109. [16] Wang, M. and Pestov, D.G. (2011) 50 -end surveillance by Xrn2 acts as a shared mechanism for mammalian pre-rRNA maturation and decay. Nucleic Acids Res. 39 (5), 1811–1822. [17] Daniely, Y. and Borowiec, J.A. (2000) Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication. J. Cell Biol. 149 (4), 799–810. [18] Wang, Y., Guan, J., Wang, H., Wang, Y., Leeper, D. and Iliakis, G. (2001) Regulation of DNA replication after heat shock by replication protein Anucleolin interactions. J. Biol. Chem. 276 (23), 20579–20588. [19] Miyagawa, R., Mizuno, R., Watanabe, K. and Ijiri, K. (2012) Formation of tRNA granules in the nucleus of heat-induced human cells. Biochem. Biophys. Res. Commun. 418, 149–155. [20] Hartford, C.M. and Ratain, M.J. (2007) Rapamycin: something old, something new, sometimes borrowed and now renewed. Clin. Pharmacol. Ther. 82 (4), 381–388. [21] Li, S., Wang, L., Berman, M., Kong, Y.Y. and Dorf, M.E. (2011) Mapping a dynamic innate immunity protein interaction network regulating type I interferon production. Immunity 35 (3), 426–440.
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