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. 2013 Sep 15;126(18):4308–4319. doi: 10.1242/jcs.134551

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival

Elio Pizzo 1,*, Carmen Sarcinelli 1,2,*, Jinghao Sheng 2, Sabato Fusco 3, Fabio Formiggini 3, Paolo Netti 3, Wenhao Yu 2, Giuseppe D'Alessio 1,‡, Guo-fu Hu 2,‡
PMCID: PMC3772394  PMID: 23843625

Summary

Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production. By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.

Key words: Angiogenin, RNH1, Stress granules

Introduction

ANG is the fifth member of the vertebrate-specific, secreted ribonuclease (RNase) family (Riordan, 2001). ANG expression is upregulated in various types of human cancer (Tello-Montoliu et al., 2006). It promotes cancer progression (Yoshioka et al., 2006) by stimulating both tumor angiogenesis (Kishimoto et al., 2005) and cancer cell growth (Tsuji et al., 2005). The growth-stimulating activity of ANG is mediated by its ability to promote ribosomal RNA (rRNA) transcription (Tsuji et al., 2005; Xu et al., 2002). ANG undergoes nuclear translocation in proliferating endothelial (Moroianu and Riordan, 1994), cancer (Tsuji et al., 2005) and neuronal (Thiyagarajan et al., 2012) cells, where it binds to the promoter region of rDNA (Xu et al., 2003) and stimulates rRNA transcription (Xu et al., 2002). ANG-mediated rRNA transcription is necessary for angiogenesis stimulated by a variety of angiogenic factors (Kishimoto et al., 2005). It also plays an important role for cancer cell proliferation in response to both genetic and environmental insults (Ibaragi et al., 2009; Yoshioka et al., 2006).

In contrast to its upregulation in various cancers, ANG is downregulated in amyotrophic lateral sclerosis (ALS) (McLaughlin et al., 2010), Parkinson's disease (PD) (Steidinger et al., 2011) and Alzheimer's disease (Kim and Kim, 2012). More importantly, loss-of-function mutations have been found in patients with ALS and PD (Conforti et al., 2008; Gellera et al., 2008; Greenway et al., 2006; Paubel et al., 2008; van Es et al., 2009; Wu et al., 2007). ANG is the only ‘loss-of-function’ mutated gene identified in ALS. Most of the PD-associated mutations are also predicted to be loss-of-function mutations. These genetic data clearly indicate that ANG plays a role in neuron survival, and its deficiency is a risk factor of neurodegenerative diseases (Li and Hu, 2010).

The extension of ANG's biological activity from cancer progression to neuron survival coincided with the recent discovery that ANG mediates the production of tiRNA (Emara et al., 2010; Fu et al., 2009; Ivanov et al., 2011; Yamasaki et al., 2009), which have been shown to suppress global protein translation of both capped and uncapped mRNA (Ivanov et al., 2011). However, IRES-mediated translation with weak eIF4G binding (Baird et al., 2006), a mechanism often used by anti-apoptosis and pro-survival genes, is not inhibited by tiRNA (Ivanov et al., 2011). Therefore, tiRNAs reprogram protein translation in response to stress, thereby promoting cell survival (Thompson et al., 2008). The production of tiRNA is induced by stress and is mediated by ANG (Yamasaki et al., 2009). Moreover, tiRNAs are able to stimulate the formation of stress granules (SGs) (Emara et al., 2010), cytoplasmic foci where untranslated mRNPs are transiently stored. Formation of SGs is an important mechanism by which cells reprogram protein translation to survive adverse conditions (Yamasaki and Anderson, 2008).

Cellular stresses inflicted by environmental and genetic factors are an underlying mechanism for both cancers and neurodegenerative diseases, the two pathological conditions where ANG has been shown to play a role. For example, hypoxic and oxidative stresses are a common etiology for cancer. Oxidative stress and endoplasmic reticulum (ER) stress resulting from accumulation of misfolded protein aggregates is a hallmark of neurodegenerative disease. It is therefore conceivable that ANG-mediated production of tiRNAs in response to stress results in reprogramming of protein translation, thereby promoting cell survival. However, a remaining question is how ANG activity is controlled so that it can properly stimulate rRNA transcription and tiRNA production, respectively, under growth and stress conditions. We hypothesized that differential subcellular localizations of ANG under growth and stress conditions might control the ultimate activity of ANG in producing either rRNA or tiRNA. The results presented in this paper show that ANG is localized to different cellular compartments under different conditions. Under growth conditions, ANG is mainly nuclear with a nucleolus accumulation. Under stress conditions, ANG is no longer localized to the nucleus, but is rather cytoplasmic and is accumulated in SGs.

The ribonucleolytic activity is essential for ANG to induce angiogenesis (Shapiro and Vallee, 1989). However, direct injection of ANG protein into the cytosol results in degradation of cellular RNA and kills the cells (Saxena et al., 1992; Saxena et al., 1991). These results raised another important question, that is, how the ribonucleolytic activity of ANG is controlled in various cellular compartments so that random RNA degradation is avoided. We hypothesized that RNH1, an abundant 50 kDa protein (Haigis et al., 2003) that binds ANG with a Kd of <1 fM (Lee et al., 1989), regulates the ribonucleolytic activity of ANG in different subcellular locations and under different growth conditions. We demonstrate in this study that under growth conditions, RNH1 is associated with cytoplasmic ANG but not with nuclear ANG so that nuclear ANG is enzymatically active but cytoplasmic ANG is inhibited. By contrast, under stress conditions, RNH1 is associated with nuclear ANG but not with cytoplasmic ANG so that nuclear ANG is inhibited but cytoplasmic ANG is not. Knockdown of RNH1 alters cellular localization of ANG and abolishes its pro-survival activity. Together, our results demonstrate that cellular activity of ANG is controlled both by its localization and by its association with RNH1.

Results

Differential subcellular localization of ANG and RNH1 under growth and stress conditions

The biological activity of ANG in mediating growth and the stress response is related to its ability in stimulating rRNA transcription and tiRNA production, respectively (Li and Hu, 2010; Li and Hu, 2012). Therefore, the ribonucleolytic activity of ANG is essential, and an important question is how ANG avoids the surveillance action of RNH1 that is abundant (Haigis et al., 2003) in both cytoplasm and nucleus (Furia et al., 2011) and that binds ANG with femtomolar affinity (Lee et al., 1989). To address this question, we first examined the protein levels of ANG and RNH1 in the cytoplasm and nucleus of HeLa cells under growth and stress conditions. Immunoblot analysis (Fig. 1A) showed that under growth conditions, more ANG is detected in the nuclear fraction than in the cytoplasmic fraction. Oxidative stress induced a shift of ANG distribution from the nucleus to the cytoplasm. When cells were stressed with sodium arsenite (SA), more ANG is detected in the cytoplasm than in the nucleus. Preferential localization of ANG to the nucleus and cytoplasm under growth and stress conditions is consistent with its respective role in stimulating rRNA transcription and tiRNA production under these conditions.

Fig. 1.

Fig. 1.

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Differential subcellular localization of ANG and RNH1 under growth and stress conditions. (A,B) Immunoblot analyses of ANG and RNH1 in nuclear and cytoplasmic fractions of HeLa cells cultured under growth and stress conditions. HeLa cells were cultured in normal growth medium (DMEM + 10% FBS) or treated with 0.5 mM SA at 37°C for 1 hour. Cells were fractionated and the nuclear and cytoplasmic proteins (30 µg) were analyzed for ANG (A) and RNH1 (B) by Immunoblot with affinity-purified pAb against ANG and RNH1, respectively. B23, nucleophosmin, a nuclear marker. (C,D) IF detection of ANG and RNH1 in HeLa cells cultured under growth (C) and stress (D) conditions. ANG mAb and Alexa-Fluor-555-labeled goat anti-mouse F(ab′)2 were used to stain ANG. RNH1 pAb and Alexa-Fluor-488-labeled goat-anti-rabbit F(ab′)2 were used to stain RNH1. Nuclei are stained with DAPI. Arrows indicate overlapping signals of ANG and RNH1. Arrowheads indicate ANG signals non-overlapping with RNH1. Dashed arrows indicate nucleoli. Scale bars: 10 µm.

The subcellular distribution pattern of RNH1 is opposite to that of ANG. More RNH1 was detected in the cytoplasmic fraction than in the nuclear fraction under growth conditions, whereas under stress conditions, more RNH1 was detected in the nucleus than in the cytoplasm (Fig. 1B).

Immunofluorescence (IF) was used to reveal more details of the converse regulation of ANG and RNH1 in the cytoplasm and nucleus under growth and stress conditions. Consistent with immunoblot results, ANG was mainly detected in the nucleus (Fig. 1C, indicated by arrows) when cells were cultured under normal growth conditions. No exogenous ANG was added to the cells in these experiments so all the IF signals were generated by endogenous ANG. Endogenous ANG was concentrated in the perinucleolar regions where rRNA processing and assembly takes place (Nazar, 2004). ANG was also detected in the cytoplasm, albeit not as strongly as in the nucleus. If exogenous ANG was added to the cells cultured under normal growth conditions, much more prominent and clear nucleolar accumulation of ANG was detected (supplementary material Fig. S1).

Under growth conditions, RNH1 was strongly detected in the nuclear plasma but not in the nucleolus (Fig. 1C, nucleoli indicated with dashed arrows). Cytoplasmic RNH1 was also visible but was not as strong as in the nucleus. The merged image shows that ANG and RNH1 are mainly colocalized in cytoplasm and nucleoplasm, but clearly not in the nucleolus. It is thus clear that under growth conditions, at least in the nucleolus, ANG is not associated with RNH1 and is not inhibited, so that nucleolar ANG remains active as a ribonuclease for the task of stimulating rRNA transcription (Xu et al., 2002).

Oxidative stress induced more cytoplasmic localization of ANG and more nuclear accumulation of RNH1 (Fig. 1D). The cytoplasmic ANG displayed a more punctate staining pattern in stressed cells. Two types of punctate cytoplasmic ANG staining were identified from the merged images: those colocalized with RNH1 (indicated by arrows) and those free of RNH1 (indicated by arrowheads). Prominent nucleolar staining of RNH1 was observed (dashed arrows), suggesting that any remaining ANG in the nucleolus would have been inhibited by RNH1.

Taken together, these results demonstrated that subcellular localization of ANG and RNH1 are dependent on the growth status of the cells and are oppositely regulated by stress. When cells are under growth conditions, ANG is mainly in the nucleus or nucleolus where it is not colocalized with RNH1 and is therefore not inhibited by RNH1 so that ANG remains fully active to stimulate rRNA transcription. When cells are stressed, the majority of ANG is not in the nucleus but RNH1 is accumulated in the nucleolus so that the trace amounts of nuclear ANG are probably inhibited by RNH1 to ensure no ANG-stimulated rRNA transcription takes place to save anabolic energy and to allocate as many resources as possible for damage repair.

Identical results were obtained with LNCaP human prostate cancer cells (supplementary material Fig. S2). Treatment with SA induced relocation of ANG from nucleus to cytoplasm and, conversely, RNH1 from cytoplasm to nucleus in LNCaP cells. Moreover, a similar pattern of relocalization of ANG and RNH1 was observed in HeLa cells treated with tunicamycin, an ER stress inducer (supplementary material Fig. S3). These results indicate that the opposite traffic of ANG and RNH1 between the nucleus and cytoplasm when the environment of the cells is shifted from growth to stress conditions is not limited to certain types of cells or stresses. It might be a general phenomenon that when cells are stressed, less ANG but more RNH1 is accumulated in the nucleus. Given the fact that a major role of nuclear ANG is to stimulate rRNA transcription, it makes sense that when cells are in conditions unfavorable for growth, ANG leaves the nucleus so that the rate of rRNA transcription is reduced to avoid energy waste for cells to survive under adverse conditions. It also makes sense for more RNH1 to accumulate in the nucleolus to ensure that the remaining ANG in the nucleolus is inhibited to halt rRNA transcription.

ANG is associated with RNH1 in cytoplasm under growth conditions and in the nucleus under stress conditions

The ribonucleolytic activity of ANG is essential for its biological activity. However, if it is not well controlled, it will randomly degrade cellular RNAs and be detrimental to the cell. In order to know whether the ribonucleolytic activity of cytoplasmic and nuclear ANG is regulated by association with RNH1 under growth and stress conditions, we performed co-immunoprecipitation (co-IP) experiments to examine the interactions between ANG and RNH1 in cytoplasmic and nuclear extracts of the cells cultured under growth and SA-induced stress conditions. RNH1 was precipitated by ANG monoclonal antibody (mAb) (Fig. 2A, lane 3). Similarly, ANG could be precipitated by RNH1 polyclonal antibody (pAb) (Fig. 2A, lane 4). Under these conditions, no ANG and RNH1 could be co-immunoprecipitated in the nuclear extracts (Fig. 2A, lanes 5 to 8). When cells were stressed with SA treatment, no association between ANG and RNH1 was detected by co-IP in the cytoplasmic extracts (Fig. 2B, lanes 1 to 4) but was apparent in the nuclear extracts (Fig. 2B, lanes 5 to 8). These results indicate that ANG is not associated with RNH1 in the nucleus when cells are cultured under growth conditions so that its ribonucleolytic activity is not inhibited by RNH1 and ANG is able to stimulate rRNA transcription thereby promoting cell growth. At the same time, cytoplasmic ANG is associated with RNH1 and its ribonucleolytic activity is inhibited to avoid RNA degradation, which would be otherwise harmful to the cell. Further, these results indicate that when cells are stressed, ANG not only moves out of the nucleus (Fig. 1) but also the remaining nuclear ANG is associated with RNH1 to ensure any trace amount of ANG activity is inhibited. Under stress condition, no association between ANG and RNH1 was detected in the cytoplasmic extracts suggesting that ANG should be active. This is consistent with the proposed function of cytoplasmic ANG in promoting cell survival by mediating the production of tiRNA under stress (Emara et al., 2010; Ivanov et al., 2011). From the viewpoint of cell survival under stress, it is reasonable that nuclear ANG is inhibited by RNH1 so that no unwanted rRNA transcription takes place to apportion as much energy as possible for damage repair and cell survival.

Fig. 2.

Fig. 2.

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Interaction between ANG and RNH1 in the nucleus and cytoplasm under growth and stress conditions. HeLa cells were cultured under normal growth conditions (A) and SA (0.5 mM, 1 hour)-induced oxidative stress conditions (B). Cells were fractionated and the cytoplasmic (lanes 1–4) and nuclear fractions (lanes 5–8) were precipitated with a non-immune mouse IgG, ANG mAb, or affinity-purified RNH1 pAb. Immunoblot analyses were performed with RNH1 pAb or ANG pAb. Input control lanes had 10% of the materials used for co-IP. The band above the specific RNH1 band is the heavy chain of the rabbit IgG. The purity of cytoplasmic and nuclear fractions was analyzed by Immunoblot analyses of β-tubulin and PCNA from 1% of the material used for co-IP.

Stress induces redistribution of cytoplasmic ANG and RNH1 into high molecular mass supracomplexes

Fig. 1 shows punctate cytoplasmic staining of both ANG and RNH1 in stressed cells, suggesting that ANG and RNH1 localizes to some cytoplasmic organelles or assembles into some supermolecular structures. We first used gel filtration chromatography on a Superdex G-200 column to examine the elution profiles of ANG and RNH1 from cytoplasmic extracts of cells cultured under growth (Fig. 3A,B) and stress (Fig. 3C,D) conditions. Oxidative stress induced a drastic shift of absorbance to the high molecular mass fractions, indicating formation of high molecular mass complexes. Accompanied with the formation of supramolecular structures, there was a shift of distribution of ANG and RNH1 in different fractions. ANG was detected only in fraction 7 and RNH1 was detected in fractions 6 and 7 (Fig. 3B) under growth conditions indicating that ANG and RNH1 existed as monomeric forms in the cytoplasm. In the fractions generated from the cytoplasm of stressed cells, ANG was detected across the entire spectrum from fractions 2 to 7 and RNH1 from fractions 2 to 6, suggesting a widespread distribution of both proteins in various structures of high molecular mass (Fig. 3D).

Fig. 3.

Fig. 3.

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Oxidative stress induces assemble of cytosolic ANG and RNH1 into high molecular mass complexes. HeLa cells were cultured under normal growth conditions (A,B) or under oxidative stress induced by 0.5 mM SA for 1 hour (C,D). Cytosolic fractions were extracted and subjected to gel filtration chromatography on a Superdex G-200 column (30 cm×1 cm, 25 ml). The elution profiles were recorded as milli absorbance units at 260 nm (A,C). The arrows indicate the elution volume of size markers, which include Herceptin (148 kDa), a compact antibody (105 kDa), ovalbumin (45 kDa) and RNaseA (15 kDa). (B,D), selected fractions from the eluates (indicated by numbers in A and C) were analyzed for ANG and RNH1 by immunoblot. Data shown are from a representative experiment of four repeats. Identical results were obtained from each experiment.

Stress-induced assembly of ANG and RNH1 in SGs

ANG has been shown to potentiate stress-induced formation of SGs through production of tiRNA (Emara et al., 2010; Yamasaki et al., 2009). However, it is unknown whether ANG itself is located in SGs and where tiRNA is produced. The findings that stresses induce relocation of cytoplasmic ANG and RNH1 to high molecular mass structures (Fig. 3), and that ANG and RNH1 display punctate staining patterns in the cytoplasm under stress (Fig. 1), led us to examine whether ANG and RNH1 are located in SGs under stress. Double IF on SA-stressed HeLa cells with an affinity-purified pAb specific to ANG and a mAb specific to polyadenine binding protein (PABP), a marker of SG (Buchan and Parker, 2009), showed colocalization of ANG and PABP (Fig. 4A, arrows), indicating that ANG is localized to SGs. It is also notable that some of the punctate ANG staining in the cytoplasm did not colocalize with PABP (Fig. 4A, arrowheads), indicating that ANG is also present in cytoplasmic organelles that are not SGs. RNH1 was also found to be present in both SGs (Fig. 4B, arrows) and non-SG organelles (Fig. 4B, arrowheads). These results demonstrated that oxidative stresses induce localization of both ANG and RNH1 in SGs. It is interesting to note that all the PABP-containing SGs (red) overlapped with ANG (Fig. 4A, green), but not all of them overlapped with RNH1 (Fig. 4B, dashed arrows). There were no SGs that did not contain ANG but there are SGs that were free of RNH1, indicating that ANG and RNH1 were colocalized in some but not all SGs.

Fig. 4.

Fig. 4.

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Detection of ANG and RNH1 in SGs. (A) Double IF of ANG and PABP. HeLa cells were incubated with 0.5 mM SA for 1 hour and were fixed by methanol at −20°C for 10 minutes. Cell nuclei were stained with DAPI. Arrows indicate ANG signals in SG. Arrowheads indicate staining of ANG in cytoplasmic organelles that are not SGs. (B) Double IF of RNH1 and PABP. Arrows and arrowheads indicate RNH1 signals in SG and non-SG cytoplasmic organelles. (C–F) Confocal image of double IF between ANG and PABP (C), RNH1 and PABP (D), ANG and TIA1 (E), and RNH1 and TIA1 (F). HeLa cells were incubated with 0.5 mM SA for 1 hour, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 (C) and (D) or fixed in methanol at −20°C (E,F). A series of Z-section images were taken and the center panel was selected for analysis. Arrows indicate SGs that were stained both for ANG and PABP (C) or TIA1 (E), and by RNH1 and PABP (D) or TIA1 (F). Dashed arrows indicate SGs that contain only PABP (A,D) or TIA1 (E,F). Images in C and D were taken with SP5 Leica confocal microscope. Images in E and F were taken with a Zeiss LSM 410 confocal microscope. Scale bars: 10 µm.

Confocal microscopy also showed that ANG was present in all SGs (Fig. 4C) but RNH1 was only found in some of the SGs (Fig. 4D). In the two cells located in a selected z-section in Fig. 4C, only 1 of the 35 countable SGs did not contain ANG (dashed arrows). The remaining 34 SGs all contained ANG (arrows), representing a 97% ANG coverage. However, only 8 of the 24 countable SGs in Fig. 4D contained RNH1 (arrows), representing an RNH1 presence in 33% of SGs. The remaining 24 SGs (67%) were RNH1 free. These results suggested that although both ANG and RNH1 are located in SGs when cells are stressed, the extent of their SG occupancy is different. These results suggest that some of the cytoplasmic ANG under stress will not be associated with RNH, consistent with our finding that ANG and RNH1 were not co-immunoprecipitated in the cytoplasmic extracts from stressed cells (Fig. 2). Very similar results were obtained with LNCaP cells (supplementary material Fig. S4), suggesting that localization of ANG and RNH1 in SGs is a general phenomenon and is not limited to a certain cell type.

Localization of ANG and RNH1 in SGs was confirmed by their colocalization with TIA1, another SG marker (Anderson and Kedersha, 2009). Granules that were both positive and negative for ANG and RNH1 were observed in TIA1-positive SGs. Again, the percentage of RNH1-positive SGs (Fig. 4F, indicated by arrows) was lower than that of ANG-positive SGs (Fig. 4E, indicated by arrows).

Fluorescence resonance energy transfer between cytoplasmic ANG and RNH1 under growth and stress conditions

Localization of both ANG and RNH1 in SGs prompted us to examine whether they are physically associated in SGs. Fluorescence resonance energy transfer (FRET) was used for this purpose. When cells were not stressed (Fig. 5A), in the marked region of interest in the cytoplasm, the intensity of green fluorescence from the donor (ANG) was 29.3±7.2 units and the intensity of the red fluorescence from the acceptor (RNH1) was 76.4±3.5 units, respectively. After photobleaching of red fluorescence to 5.3±0.4 units (6.9% of the original), the intensity of the donor fluorescence became 32.0±7.6 units, representing an increase of 10.1±1.2%. Therefore, there was an energy transfer from donor fluorophore to acceptor fluorophore suggesting that a physical interaction existed between ANG and RNH1 in the cytoplasm when cells were cultured under normal growth conditions. In the selected SGs that contained both ANG and RNH1, no energy transfer was observed (Fig. 5B). The fluorescence intensity of the donor was 75.4±9.1 and 76.0±9.6 units, respectively, before and after photobleaching of the acceptor, representing a mere 0.8% increase. These results indicated that ANG and RNH1 were not physically associated in SGs even though they were both located there. Therefore, cytoplasmic ANG retains its ribonucleolytic activity for the purpose of generating tiRNA to promote cell survival under stress.

Fig. 5.

Fig. 5.

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Immuno-FRET between cytoplasmic ANG and RNH1 under growth and stress conditions. HeLa cells were cultured in normal growth conditions (A) or treated with 0.5 mM SA for 1 hour (B), fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. ANG and RNH1 were detected with mAb and pAb, respectively, and visualized with Alexa-Fluor-488- and Cy3-labeled secondary antibodies. Green and red fluorescence confocal images were taken under a single excitation (488 nm) wavelength and were merged. The center panel of the Z-section images was used for FRET analyses. The intensity of both green (donor, ANG) and red (acceptor, RNH1) fluorescence in the selected ROI was recorded. The red fluorescence was then bleached at 592 nm for 5 seconds, and the green and red fluorescence was recorded again. Scale bars: 5 µm.

Localization of ANG and RNH1 in cells recovered from stress

HeLa cells were stressed with 0.5 mM SA for 1 hour and then allowed to recover in full growth medium for 3 hours. Subcellular localization of ANG and RNH1 in recovered cells was first examined by double IF (supplementary material Fig. S5A) and was found to be very similar to that of the cells cultured only in growth conditions (Fig. 1C). The most significant feature is that ANG relocated back to the nucleus and that RNH1 disappeared from nucleolus (supplementary material Fig. S5A, dashed arrows). There was still a trace amount of both ANG and RNH1 in SGs (supplementary material Fig. S5B,C) but both the number and the size of SGs was significantly reduced compared with that in the stressed cells (Fig. 4). These results indicate that when cells were recovered from stress, SGs dissembled and therefore ANG and RNH1 relocated both to the cytoplasm and nucleus. Specifically, RNH1 disappeared from the nucleolus so that the ribonucleolytic activity of ANG in the nucleolus resumed, enabling its activity in stimulating rRNA transcription.

The fate of cytoplasmic ANG and RNH1 in recovered cells was further studied by using a sucrose gradient. Fig. 6A shows the sucrose gradient profiles of the cytoplasmic extracts of cells that have been cultured in growth, stress and stress recovery conditions. Similar to the results obtained from gel filtration chromatography, the most significant difference between growth and stress conditions was noticed in the high molecular mass region between fractions 3 and 9 where the UV absorbance was much higher in the fractions generated from stressed cells. The profile from stress-recovered cells was very similar to that from the cells cultured under growth conditions. Fig. 6B is the distribution of cellular RNA (mainly rRNA) in the sucrose gradient fractions, which marks the region (fractions 3–9) where ribosomes were eluted. It is notable that total RNA is decreased in the stressed cells, in agreement with an early finding that stress decreases the abundance of polyribosomes (Bevilacqua et al., 2010). Because the size of SGs and ribosomes are similar (Souquere et al., 2009), it is likely that SGs were also distributed in these fractions. This hypothesis was confirmed by immunoblot analysis of PABP (Fig. 6C). Under growth conditions, PABP was detected in a roughly equal density in every fraction, indicating an even distribution in the cytoplasm. In stressed cells, PABP were enriched in fractions 3–11, indicating that it was these fractions where SGs were eluted. Both ANG and RNH1 were found in the lower molecular mass fractions under growth conditions, confirming that cytoplasmic ANG and RNH1 were not in supramolecular structures. Stress-induced relocation of both ANG and RNH1 to high molecular mass sucrose gradient fractions was confirmed. It is of significant interest to note that ANG and RNH1 were detected in lower molecular mass fractions in the cells recovered from stress (Fig. 6C, bottom three panels), indicating that a recovery process of cells from stress is accompanied with a return of ANG and RNH1 to their normal localization patterns. These results indicate that stress-induced relocalization of ANG and RNH1 is dynamic and is restored when stress is lifted.

Fig. 6.

Fig. 6.

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Dynamic localization of cytoplasmic ANG and RNH1 under various growth conditions. HeLa cells were cultured under growth and oxidative stress conditions (0.5 mM SA, 1 hour). In the recovery experiment, SA was removed by medium exchange and the cells were washed three times and cultured in growth conditions for 3 hours. Cells were fractionated and the cytoplasmic fractions were ultracentrifuged in a sucrose gradient (60–15%). (A) Absorbance profiles at 260 nm of the sucrose gradient fractions. (B) RNA content in each fraction. 50 µl from every other fraction of the gradient were separated in 1% agarose gels and stained with EB. (C) Immunoblot analyses of ANG, RNH1 and PABP from alternate gradient fractions collected in A. Data shown are from a representative experiment of four repeats. Identical results were obtained from each repeat.

Knockdown of RNH1 impairs localization of ANG in SGs and alters cell growth and survival behavior

To understand the effect of RNH1 on localization of ANG to SGs when cells were exposed to stress, we used lentivirus-mediated small hairpin RNA (shRNA) to knock down RNH1 expression and examined the resultant changes in ANG localization by IF. Among the five different shRNA constructs, four of them knocked down RNH1 expression efficiently as shown in Fig. 7A. The relative intensity of RNH1 to β-tubulin in the cells infected with lentivirus encoding a scramble shRNA, RNH1-specific shRNA 33, 34, 35 and 37 were 100, 8.6±0.1, 18.2±4.8, 3.0±0.7 and 12.9±6.9, respectively. Thus, clone 35 (Sh35) was most efficient in knocking down RNH1 expression with a 97% decrease in RNH1 protein level, and was selected for further study. Scramble control and Sh35 shRNA transfected HeLa cells were stressed with SA and localization of ANG was examined by IF. ANG was found to be colocalized with PABP in SGs in scramble shRNA transfected cells (Fig. 7B), not so different from the pattern seen with untransfected HeLa cells shown in Fig. 4A. However, in RNH1-knockdown cells, SGs were formed normally but no ANG was colocalized with PABP (Fig. 7C). Instead, ANG was found in the nucleolus (indicated by dashed arrows). These results indicate that stress-induced relocalization of ANG in SGs was impaired when RNH1 was downregulated.

Fig. 7.

Fig. 7.

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Knockdown of RNH1 alters subcellular localization of ANG under stress. HeLa cells were infected with pLKO.1 lentiviral particles encoding RNH1-specific shRNA and scramble control. Infected cells were selected in the presence of 1.5 µg/ml puromycin. (A) RNH1 levels in scramble control and specific shRNA lentivirus-infected HeLa cells. Left panel, Immunoblots. Right panel, ImageJ analyses of relative band intensity of RNH1 over β-tubulin. (B,C) Subcellular localization of ANG in RNH1-knockdown cells under oxidative stress. Cells infected with scramble control (B) and Sh35 (C) lentivirus were subjected to 0.5 mM SA for 1 hour and stained for ANG and PABP. Arrows indicate colocalization of ANG and PABP in control cells. Arrowheads indicate nucleoli staining of ANG in RNH1-knockdown cells. Scale bars: 10 µm. Similar results were obtained in cells infected with Sh33 and Sh34.

Cell proliferation in RNH1-knockdown cells was significantly reduced compared with levels in the scramble control (Fig. 8A). All four shRNA constructs inhibited cell growth and the extents of inhibition correlated positively to the knockdown efficiency (Fig. 7A, Fig. 8A). We next examined the sensitivity of RNH1-knockdown cells to SA stress. Fig. 8B,C shows that RNH1 knockdown decreased viability of the cells under oxidative stress. When cells were treated with 0.5 mM SA for 4 hours, the number of viable cells in scramble and Sh35 transfectants was 57% and 26%, respectively, of that before the treatment (Fig. 8B), representing a 37% increase in cell death rate in RNH1-knockdown cells. When the SA concentration was increased to 1 mM, cell death was obvious after 1 hour of treatment (Fig. 8C). Again, RNH1-knockdown cells were more sensitive than the scramble control. Viability of scramble and Sh35 transfectants was 81% and 60%, respectively (Fig. 8C). At 2 mM SA, massive cell death occurred in both scramble and Sh35 transfectants. However, it is apparent that there were still more viable cells in control cells (15%) than in RNH1-knockdown cells (9%). Taken together, these results indicate that RNH1 knockdown changed the localization of ANG as well as cell growth and survival, suggesting that a proper RNH1 level might be essential for the cellular functions of ANG.

Fig. 8.

Fig. 8.

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Knockdown of RNH1 in HeLa cells decrease growth and increases sensitivity to stress. (A) Cell growth. Stable scramble and RNH1 shRNA transfectants were cultured in DMEM + 10% FBS in the presence of 0.5 µg/ml ANG. Cell numbers were determined with a Coulter counter. (B) Time course of cell survival. Cells were cultured in growth medium for 48 hours, and then subjected to treatment with 0.5 mM SA for the indicated time. Cell numbers were determined by MTT assay. (C) SA dose response of cell survival. Cells were cultured in growth medium for 48 hours and treated with different concentrations of SA for 1 hour. Cell numbers were determined by MTT assay. Data shown in A–C are means ± s.d. of triplicates of a representative experiment. (D) Cell apoptosis under growth conditions. Cells were cultured in DMEM + 10% FBS and stained with EB and AO. Both apoptotic (red) and live (green) cells were counted. (E) SA-induced apoptosis. Cells were treated with 1 mM SA for 1 hour and stained with EB and AO. The numbers shown in D–E are means ± s.d. of the percentage of apoptotic cells counted in five microscopic fields.

To examine the effect of RNH1 knockdown on cell apoptosis, ethidium bromide (EB) and Acridine Orange (AO) staining was used to stain apoptotic cells. AO permeates intact cells and stains all nuclei green whereas EB enters cells only when the integrity of plasma membrane is compromised so it stains apoptotic nuclei red (Ribble et al., 2005). Cell apoptosis significantly increased in RNH1-knockdown cells when they were cultured both under growth (Fig. 8D) and stress (Fig. 8E) conditions. The percentage of apoptotic cells in Sh35 transfectants was 11.8±3.7%, which is 1.9-fold that in scramble control transfectants (6.2±2.7%). When the cells were treated with 1 mM SA for 1 hour, the percentage of apoptotic cells in scramble and Sh35 transfectants was 11.3±4.5% and 15.9±4.9%, respectively, representing a 41% increase in SA-induced apoptosis when RNH1 was knocked down. These results suggest that increased apoptosis might be a reason for decreased growth and survival in RNH1-knockdown cells.

Discussion

Dynamic cellular localization of ANG and RNH1

Our results show that subcellular localization of ANG is dynamic and is dependent on the growth status of the cell. ANG is mainly localized to the nucleus when cells are in normal growth conditions. Oxidative and ER stress both induced a shift of ANG localization from nucleus to cytoplasm. Differential subcellular localization of ANG under different growth conditions might be a mechanism of regulation of the growth and survival functions of ANG. The growth-stimulating function of ANG is mediated by its activity in promoting rRNA transcription. For this purpose, ANG needs to bind to the promoter region of rDNA, which is probably the reason why ANG is mainly in the nucleus to meet the high metabolic demand of growing cells for rRNA. The pro-survival function of ANG is mediated by its ability to produce tiRNA. When cells are in adverse conditions, the protein translation rate is decreased to save anabolic energy for survival. The production of tiRNA meets this purpose because tiRNAs suppress global protein translation but do not alter IRES-mediated translations of anti-apoptotic mRNAs. It therefore makes sense for ANG to leave nucleus when cells are under stress to avoid waste of resources in producing unnecessary rRNA.

A surprising finding is that RNH1 was found to be mainly located in the nucleus under growth conditions. RNH1 was previously considered to be a cytoplasmic protein (Haigis et al., 2003) even though it had also been detected in the nucleus (Furia et al., 2011). The role of nuclear RNH1 under growth conditions is currently unclear. However, it is not associated with ANG as shown by co-IP experiments. Thus, normal function of nuclear ANG in stimulating rRNA transcription is not inhibited by RNH1 under growth conditions. At the same time, cytoplasmic ANG is associated with RNH1, as shown by both co-IP and FRET experiments. RNH1 binds to ANG with a sub-femtomolar affinity (Lee et al., 1989) and has been shown to inhibit both enzymatic and angiogenic activity of ANG. We can reasonably assume that when ANG is bound by RNH1, its activity will be completely inhibited. Therefore, under growth conditions, the small amount of ANG in the cytoplasm is most likely inhibited by RNH1 so that no unfavorable RNA degradation occurs in the cytoplasm to maintain a proper healthy status of the cell.

Even more RNH1 was found in the nucleus when cells were stressed. More significantly, prominent nucleolar localization of RNH1 was noticed. It is reasonable for nuclear RNH1 to be located in nucleolus under stress conditions so that any trace amount of ANG remained in the nucleolus will be bound and inhibited by RNH1. Indeed, co-IP results showed that ANG and RNH1 were associated in the nuclear fractions extracted from stressed cells. Stresses not only reduce the amount of cytoplasmic RNH1, but also disable its interaction with ANG in cytoplasm. From a functional point of view, it is reasonable for RNH1 to dissociate from ANG in cytoplasm under stress so that ANG regains its ribonucleolytic activity for the purpose of tiRNA production. RNH1 is sensitive to oxidation, attributable to its Cys residues. RNH1 contains 32 free Cys residues and no disulfide bonds, and loses activity in the absence of reducing agents (Blackburn et al., 1977). Treatment of RNH1–RNase complexes with p-hydroxymercuribenzoate rapidly dissociates the complex, releasing fully active RNase. Oxidation or derivatization of Cys residues, none of which forms important contacts with ANG (Papageorgiou et al., 1997), drastically alters the 3D structure of RNH1 (Fominaya and Hofsteenge, 1992), which may lead to dissociation of ANG from RNH1–ANG complex. In any event, ANG dissociates from RNH1 in cytoplasm under stress, suggesting that cytoplasm is the likely place where tiRNA is produced. These results demonstrated that interacting with RNH1 fine-tunes the regulatory activities of ANG in stimulating cell growth or in promoting cell survival.

RNH1 regulates SG localization of ANG under stress

Stress induces traffic of ANG not only from the nucleus to the cytoplasm but also from low molecular mass fractions to high molecular supramolecular structures within the cytoplasm. One class of these supramolecular structures is the SG. It is significant that ANG is located in SGs in stressed cells and that this localization is regulated by RNH1. ANG was detected in almost every SG induced by SA. RNH1 was also found in some but not all SGs. So a subset of SGs contained ANG but was RNH1 free. Moreover, in those SGs that contained both ANG and RNH1, ANG was not associated with RNH1. Therefore, cytoplasmic ANG that is localized in SGs would be enzymatically active. SGs are primarily composed of the stalled 48S preinitiation complexes containing mRNA bound to small ribosome subunits and the initiation factors (Anderson and Kedersha, 2008). SGs also contain other proteins with diverse functions including RNA-binding proteins, RNA helicases, nucleases, kinases and signaling molecules (Anderson and Kedersha, 2009). It is of particular interest to note that SGs also contain RNA-induced silencing complexes. It is therefore conceivable that the function of ANG located in SGs may be integrated with microRNA-induced translational silencing mechanism thus potentially be involved in diverse cellular pathways. No matter what role ANG plays in SGs, it is unlikely that SGs is the place where ANG cleaves tRNA. Knockdown of RNH1 drastically reduced localization of ANG in SGs. tiRNA production would be expected to be decreased in RNH1-knockdown cells if it were SGs where ANG produced tiRNA. But it has been shown that tiRNA production is increased when the RNH1 level is downregulated (Yamasaki et al., 2009).

ANG was also localized in other classes of superamolecular structure that are not SGs. The nature of these structures are unknown at present but they are probably not P-bodies because staining with P-body markers did not show colocalization with ANG. Localization of ANG to this type of structure was significantly increased in RNH1-knockdown cells when they were subjected to stress. One possible candidate for this structure(s) is the polysome or the 60S subunit where ANG has been detected. If the polysome is the place where tiRNA is produced, it will be consistent with the recent finding that tiRNA production is higher when protein synthesis is active and when tRNAs transit more frequently between the ribosome-bound and the aminoacyltRNA-synthetase-bound states (Saikia et al., 2012). In any event, these results demonstrate that both the cellular localization and function of ANG are regulated by the RNH1 level.

RNH1 regulates cell proliferation and survival

RNH1 is one of the most abundant cellular proteins accounting for 0.08% of total cytosolic protein content (Haigis et al., 2003). The biochemical properties of RNH1 have been well documented, including characterization of high-resolution X-ray structures of free RNH1 (Kobe and Deisenhofer, 1993) and in complex with RNaseA (Kobe and Deisenhofer, 1996), ANG (Papageorgiou et al., 1997) and RNase1 (Johnson et al., 2007). RNH1 is composed of seven leucine-rich repeats and a conserved structure domain that are often involved in protein–protein interactions (Kobe and Deisenhofer, 1995) and that have been found in many proteins with diverse cellular functions ranging from cell-cycle regulation to DNA repair to immune regulation (Bella et al., 2008; de Wit et al., 2011). Compared with the well-defined biochemical properties of RNH1, its physiological and pathological roles are evolving. RNH1 was first thought to serve as cellular ‘sentry’ (Haigis et al., 2003) to protect cells from damage caused by non-cytosolic RNase that gain entry into the cytoplasm. Indeed, extensive efforts have been put to design cytotoxic RNase that do not bind RNH1 for therapeutic purposes in cancer treatment (Lee and Raines, 2008; Rutkoski and Raines, 2008).

We have found that knockdown of RNH1 inhibited cell proliferation in a dose-dependent manner. One of the reasons for decreased cell growth in RNH1-knockdown cells can be attributed to increased apoptosis. The percentage of apoptotic cells doubled when RNH1 was knocked down. Knockdown of RNH1 also significantly decreased cell survival under stress conditions, accompanied with an increase in cell apoptosis. Decreased survival of RNH1-knockdown cells against oxidative stress is probably related to abnormal cellular localization of ANG. The fact that ANG is localized in the nucleolus but not in SGs when RNH1-knockdown cells were subjected to SA stress might be the reason for decreased survival. rRNA transcription is an energetically costly process. From the cell survival viewpoint, it is certainly counterproductive for ANG to be in the nucleolus to continually produce rRNA when cells are stressed. Failure of ANG to localize to SGs in stressed cells could also cause decreased survival of RNH1-knockdown cells. Thus, a major role of RNH1 is to regulate cellular localization of ANG, thereby controlling cell growth and survival. The reported antioxidant (Cui et al., 2003) and redox homeostatic (Monti et al., 2007) effects might also contribute to the regulatory function of RNH1 in cell survival. RNH1 has also been shown to mediate pri-miR-21 processing, thereby contributing to cancer progression (Kim et al., 2011). In view of the facts that ANG is a RNase and that ANG, RNH1 and the RISC complex are all found in SGs, it is tempting to speculate that ANG and RNH1 play a role in micro RNA biogenesis and that SGs are an additional or alternative place where micro RNAs are generated or metabolized.

Materials and Methods

Cell cultures and treatment

HeLa cells were maintained in DMEM supplemented with 10% FBS. LNCaP cells were maintained in RPMI 1640 supplemented with 10% FBS. To induce oxidative stress, cells were treated with 0.5 mM SA for 1 hour or as otherwise indicated. To induce ER stress, cells were treated with 5 µg/ml tunicamycin for 24 hours. In the recovery experiments, medium containing SA was removed and cells were washed with DMEM three times and cultured in growth medium for 3 hours.

Cell extracts and co-IP

Cells were detached by trypsin-EDTA and resuspended in 10 mM HEPES, pH 8.0, containing 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and 1× proteinase inhibitors cocktail and incubated on ice for 10 minutes. Cells were lysed by adding NP-40 to a final concentration of 0.1%. Cytoplasmic fraction was obtained by centrifugation at 1000g for 5 minutes. The pellet was dissolved in RIPA buffer and was designated as the nuclear fraction. The purity of the cytoplasmic and nuclear fractions was examined by immunoblot analyses of β-tubulin and PCNA, respectively. For co-IP experiments, the cytoplasmic and nuclear fractions from 4×106 cells were diluted in 1 ml of 10 mM HEPES containing 0.1% NP-40. A fraction of 50 µl was taken and used as input control. The remaining materials were divided into three equal fractions, incubated with 5 µg of non-immune mouse IgG, ANG mAb 26-2F, or affinity-purified RNH1 pAb R127 at 4°C. Five µl from each sample was taken for β-tubulin and PCNA analysis. The remaining solution was mixed with 50 µl of 50% Protein A/G-Sepharose by rotating at 4°C for 2 hours. The mixture was centrifuged, washed and analysed by Immunoblot for ANG and RNH1 with R113 and R127, respectively.

Gel filtration chromatography

The cytoplasmic fraction from 6×106 HeLa cells was diluted in 200 µl of PBS and applied to a Superdex G-200 column (30 cm×1 cm, 25 ml) equilibrated in PBS. The eluate was monitored at 260 nm, collected in 0.25 ml fractions and analyzed for ANG and RNH1 contents by Immunoblot.

Sucrose gradient ultracentrifugation

The cytoplasmic fractions of the cells were layered onto 60–15% sucrose gradients (10 mM HEPES, pH 7.4, 5 mM MgCl2 and 300 mM KCl). After centrifugation at 38,000 r.p.m. overnight at 4°C in a Beckman SW 41 rotor, the gradient fractions were collected from the bottom of the tubes using a peristaltic pump and monitored by UV absorbance at 260 nm. RNA contents were analyzed by agarose gel electrophoresis. ANG and RNH1 were analyzed by Immunoblot.

Immunofluorescence and confocal microscopy

Cells were cultured on coverslips and were fixed in methanol at −20°C for 10 minutes, blocked with 3% BSA in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% Tween 20. The antibodies used were ANG mAb 26-2F (5 µg/ml), affinity-purified ANG pAb R113 (2 µg/ml), affinity-purified RNH1 pAb R127 (2 µg/ml), PABP mAb 10E10 (Abcam, cat #ab6125, 1 µg/ml) and TIA1 mAb (Abcam, cat #ab2712, 1 µg/ml). The secondary antibodies used were Alexa-Fluor-488-conjugated goat anti-rabbit or Alexa-Fluor-555-conjugated goat anti-mouse F(ab′)2 (1∶1000 dilution). Confocal microscopy was performed with a confocal laser-scanner microscope SP5 Leica and by Zeiss LSM 410. For SP5 Leica, the lambda of the argon ion laser and HeNe laser was set at 488 nm and 546 nm, respectively. Fluorescence emission was revealed by band pass 500–530 and 560–650, respectively, for Alexa Fluor 488 and Alexa Fluor 546. For Zeiss LSM 410, ArKr Laser (488/568/647) was used. Band pass 510–525 and 590 and 610 was used for Alexa Fluor 488 and Alexa Fluor 555.

FRET

The acceptor photo-bleaching method was used. The donor (Alexa Fluor 488) was excited at 488 nm and detected at 500–530 nm. The acceptor (Cy3) bleaching was performed at 592 nm. FRET was measured by the increase of Alexa Fluor 488 fluorescence intensity after Cy3 photo-bleaching. Measurements were performed on ROI in cytoplasm (n = 10) under growth and in SG under stress (n = 7) conditions. To ensure reproducibility and reliability of Alexa Fluor 488 fluorescence measurements, Cy3 was photo-bleached to <10% of its initial fluorescence. Efficiency of FRET was calculated as E = (IDA−ID)/ID where ID and IDA are fluorescence intensities before and after photo-bleaching.

RNH1 knockdown

A set of human RNH1-specific shRNA cloned in pLKO.1 lentiviral vector was purchased from Open Biosystems. Lentiviral particles were packaged in HEK293T cells co-transfected with shRNA inserted pLKO.1 (5.8 µg), envelope plasmid pMD2.G (1.8 µg) and packaging plasmid psPAX (4.4 µg). HeLa cells were infected with lentivirus in the presence of 10 µg/ml polybrene for 48 hours. Puromycin-resistant cells were selected and the level of RNH1 was examined by Immunoblot analysis.

EB and AO staining of apoptotic cells

Apoptotic cells were identified by EB and AO staining as described (Ribble et al., 2005). Cells before or after SA treatment (1 mM for 1 hour) were trypsinized, pelleted and washed with 4°C PBS. The cells were resuspended in 100 µl of 4°C PBS and mixed with 5 µl of the EB/AO dye mixture (100 µg/ml each of AO and EB in PBS) at 37°C for 20 minutes. Stained cells were placed on a microscope slide and covered with coverslips.

Supplementary Material

Supplementary Material
supp_126_18_4308__index.html (915B, html)

Acknowledgments

We thank Daria M. Monti, Rita Del Giudice, Giuseppina Fusco and Hailing Yang for helpful discussions and technical assistance.

Footnotes

Author contributions

E.P., C.S., J.S., S.F., F.F., P.N. and E.Y. performed experiments. E.P., G.D. and G.-f.H. conceived ideas, analyzed and interpreted results. G.-f.H. wrote the manuscript.

Funding

This work was supported in part by Italian Ministry of University and by the National Institutes of Health [grant numbers R01 NS065237 and R01 CA105241 to G.-f.H.]. Deposited in PMC for release after 12 months.

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