Oncogene (2009) 28, 1782–1791 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE RNase L downmodulation of the RNA-binding protein, HuR, and cellular growth

W Al-Ahmadi1, L al-Haj1, FA Al-Mohanna1, RH Silverman2 and KSA Khabar1,2

1Program in BioMolecular Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia and 2Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, OH, USA

Ribonuclease L (RNase L) is an intracellular that vision (ELAV) proteins comprising a group of is vital in innate immunity, but also is a tumor suppressor RNA-binding proteins first described in Drosophila. candidate. Here, we show that overexpression of RNase L HuR is implicated in the stabilization of several decreases cellular growth and downmodulates the RNA- adenylate uridylate (AU)-rich mRNAs including those binding protein, HuR, a regulator of -cycle progression that regulate the cell cycle such as cyclins A and B, and tumorigenesis. The effect is temporal, occurring in and p21, and those that affect tumor activities specific cell-cycle phases and correlated with the cyto- such as urokinase activator (uPA) and cyclooxygenase, plasmic localization of RNase L. Both cellular growth and COX-2 (Wang et al., 2000, 2001; Subbaramaiah HuR were increased in RNASEL-null mouse fibroblast et al., 2003; Tran et al., 2003). Knockout of HuR by lines when compared to wild-type cells. Moreover, the siRNA or antisense vectors decreases cellular growth stability of HuR mRNA was enhanced in RNASEL-null (Lopez de Silanes et al., 2003; Dormoy-Raclet et al., cells. The HuR 30 untranslated region (UTR), which 2007). harbors U-rich and adenylate-uridylate-rich elements, was RNase L is activated by 20,50-oligoadenylates (2-5A), potently responsive to RNase L when compared to control which are synthesized from ATP by the 20,50-oligoade- 30 UTR. Our results may offer a new explanation to the nylate synthetase (OAS), an (IFN)-inducible tumor suppressor function of RNase L. product. OAS itself is expressed as a latent protein Oncogene (2009) 28, 1782–1791; doi:10.1038/onc.2009.16; that is active on binding to double-stranded RNA. The published online 2 March 2009 latter is produced as an intermediate during the viral life cycle (Stark et al., 1998) or possibly due to cellular RNA Keywords: 30 untranslated regions; RNase L; HuR; with double-stranded structures (Wang and Carmichael, RNA-binding proteins; cell growth 2004; Molinaro et al., 2006). RNase L has been known for its involvement in the defense against viruses due to its potent degrading effect on viral mRNAs (Kumar et al., 1988; Zhou et al., 1997, 1998; Li et al., 1998; Khabar et al., 2000). Moreover, it has been shown that Introduction subjects with mutations in RNASEL are more susceptible to infection by a novel gammaretro- Ribonuclease L (RNase L) is an intracellular endor- virus (Urisman et al., 2006). ibonuclease that has been suggested as a tumor RNase L, itself, does not cause global degradation of suppressor candidate due to linkage with hereditary cellular mRNA in intact cells due to the low endogenous prostate cancer (HPC1) gene and because of its levels of 2-5A. Rather, 2-5A at high concentrations leads proapoptotic role (Zhou et al., 1998; Carpten et al., to degradation of ribosomal RNA, such as those 2002; Silverman, 2003). The mechanism of RNase L on encountered during acute viral replication or when cell division is not known. Classically, many tumor exogenously added to cells (Wreschner et al., 1981a; suppressors, such as p53, halt cell-cycle progression by Cirino et al., 1997; Li et al., 1998). Previously, we were regulating cell-cycle check points governed by cyclins able to provide the first evidence that RNase L, in the and cyclin-dependent kinases. But there are proteins of absence of viral infection, can act on cellular mRNAs different functional classes that affect these activities (Khabar et al., 1999, 2003). Specifically, RNase L such as the RNA-binding protein, HuR, a member of downregulates the double-stranded RNA-dependent mammalian homologues of embryonic lethal abnormal protein kinase (PKR) mRNA and subsequently leads to downregulation of the consequences of this effect, that is, eIF2a phosphorylation and inhibition of protein Correspondence: Dr KSA Khabar, Program in BioMolecular Re- synthesis (Khabar et al., 2003). Here, our findings search, King Faisal Specialist Hospital and Research Center, P3354, revealed that RNase L downmodulates HuR mRNA MBC-03, Takhasusi Road, Riyadh 11211, Saudi Arabia. and subsequently HuR protein in a manner that is E-mail: [email protected] Received 5 August 2008; revised 13 November 2008; accepted 1 correlated with RNase-L-induced suppression of cellu- December 2008; published online 2 March 2009 lar growth and cytoplasmic localization of RNase L. RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1783 Results We used a new continuous real-time biosensor that measures cellular growth based on impedance (Solly Moderate overexpression of RNase L and effects et al., 2004; Zhu et al., 2006), to assess the effect of on cellular growth RNase L expression. The RNase-L-expressing cell line To elucidate the mechanism of the cell growth suppres- had reduced growth rates when compared to the control sor action of RNase L, we generated a polyclonal cell cell line and was more pronounced when cells were line from a HeLa line—with undetectable or very low approaching confluence (Figure 1a). These results were expression of RNase L—that stably expresses moderate similar when a colorimetric conventional assay (Alamar amounts of RNase L. Approximately twofold expres- blue) was used (data not shown). The antiproliferative sion in RNase L at the protein level when compared to effect observed was not due to as the vector was achieved (Supplementary Figure 1). We proapoptotic effect of RNase L requires an apoptosis chose a short-term polyclonal line generation approach inducer (Diaz-Guerra et al., 1997; Zhou et al., 1997; in contrast to monoclonal lines to avoid cell selection Castelli et al., 1998), which was not used in this study. bias as subtle variations in RNase L can lead to Also, flow cytometry analysis that measures DNA significant changes in mRNA levels. content of sub-G1 cells showed there was no difference

AU-Rich Microarray Profiling 3.5

3.0 145 30 Neo 2.5 RNase L Down- HuR Up- 2.0 p<0.001 Regulated Regulated 1.5

CELL INDEX -4 +4 1.0

0.5 Neo/RNase L HuR RNAse L HeLa-HuRHeLa-Vector 0.0 HUR (ELAV1) 0 20 40 60 80 100 120 plasminogen activator HuR Cyclin D1 (CCND1) TIME, HR GAPDH (GAPD) GAPDH Beta Actin (ACTB) Ribosomal Protein S25 (RPS25)

**

100

NEO NEO/ RNASE L 80 HuR 60

VECTOR/NEORNASE L/NEO β-ACTIN 40 HUR 36kDa normalized 20 β-ACTIN 40kDa 0 % of control (neo vector), Neo Neo/RNase L Figure 1 RNase L effect on cellular growth and cellular mRNAs. (a) Growth curves of RNase-L-expressing and empty vector polyclonal cells. RNase L/neomycin or vector/neomycin cells were seeded 5  103 cells per well in 96-well plates and then monitored for growth by continuous real-time RT-CES biosensor each 20 min for a period of 5 days. Data are from four replicates (shaded gray areas are s.e.m.). Curve fit and doubling time was assessed using the exponential growth equation Y ¼ Start  exp(K  X); Y, start and increases exponentially with rate constant K; the doubling time equals 0.69/K. Curve comparison (K parameter difference) was used using F-test (P-values are indicated). (b) Microarray profiling of AU-rich mRNA expression as a result of RNase L and HuR action. Colored list shows the that are related in this study, which are significant and statistically reproducible between control and either RNase-L-expressing or HuR-overexpressing (HeLa) lines. The color legend shows gradation of color proportional to the magnitude of induction (red) or repression (green) of expression. Inside image is a representative western blot for the HuR-overexpressing cell line. (c) Total RNA was extracted from confluent cells and used for RT–PCR at semi-quantitative conditions as described in Materials and methods, with primers specific to HuR and b-actin mRNA. (d) Equal amount of proteins extracted from confluent cells that stably express neomycin vector or RNase L/neomycin was analysed by western blotting using anti-HuR and anti-b-actin. Data shown are two representative experiments of three. (Right) Quantitation of b-actin-normalized signals (mean±s.e.m.) of HuR from three independent experiments. **Po0.01 using Student’s t-test statistic.

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1784 in the percentage of apoptotic cells; o3% of the total mRNA levels in the same cell type (Figure 1b). Because cell pool in both vector and RNase-L-expressing the microarray data were for screening purposes, we cell lines. further focused on HuR rather than patterns of the individual genes in the array data. Semi- quantitative RT–PCR experiments confirmed that Cellular mRNA changes due to RNase L RNase L led to reduction in HuR mRNA levels To find which gene products affect cellular growth and (Figure 1c). To confirm the findings that RNase L led are modulated by RNase L, we used custom-made to HuR downregulation, we measured levels of HuR microarrays (Frevel et al., 2003; Khabar et al., 2004) protein by western blotting. Levels of HuR protein that contain probes for U-rich mRNAs—RNase L is were reduced (B2-fold reduction, P 0.01) in RNase-L- known to cleave at U-rich dinucleotides (Wreschner o expressing cells when compared to control cells et al., 1981b). Comparing microarray gene expression (Figure 1d). patterns from cells expressing RNase L with control vector revealed that the mRNA levels of the RNA- binding protein, HuR, were reduced in RNase L HeLa- Effect of RNase L and HuR knockdown on HuR protein expressing cells (Figure 1b). In addition, two known and cellular growth mRNA targets for HuR, urokinase plasminogen acti- Further, we used mouse embryonic fibroblast (MEFs) vator (uPA) and cyclin D1 (Tran et al., 2003; Lal et al., lines generated from RNASEL-knockout and wild-type 2004), were reduced in RNase-L-expressing HeLa mice (Zhou et al., 1997). Western blotting confirmed cells. As a control, HeLa cells overexpressing HuR the absence of RNase L using anti-mouse RNase L (Figure 1b, inset blot) have increased uPA and cyclin D1 antibody (Figure 2a). Increased HuR levels (2.5-fold,

-/- 10.0

RNase L-/- RNAse LRNAse +/+ L p<0.001 RNAse L 85 kDa 7.5

HuR 36 kDa 5.0 42 kDa β-ACTIN CELL INDEX +/+ *** 2.5 RNase L 280 240 0.0 200 01020 30 40 50 60 70 80 90 100 110 120 160 TIME, HR 120 normalized 80 1.50 40

% of control (wild-type), RNase L shRNA HuR 0 1.25 Vector/shRNA HuR RNase L+/+ RNase L-/-

1.00 Vector/shRNA Ctrl -/- -/-

0.75 RNAse LRNAse +/+ LRNAse LRNAse +/+ L

IFN − − ++ Cell Index 0.50 RNase L/ shRNA Ctrl RNAse L 85 kDa 0.25 HuR 36 kDa 0.00 GAPDH 40 kDa 0 102030405060708090100 Time, HR Figure 2 Effect of RNase L overexpression and knockout on HuR and cellular growth. (a) Equal amount of proteins extracted from confluent mouse embryonic fibroblasts (MEFs) generated from wild-type or RNASEL-knockout mice was used for western blotting (anti-mouse RNase L, anti-HuR and anti-b-actin). (Lower panel) Quantitation of b-actin-normalized signals (mean±s.e.m.) of HuR from three independent experiments. ***Po0.001 using Student’s t-test statistics. (b) Confluent MEFs generated from wild-type or RNASEL-knockout mice were treated for 16 h with mock or interferon-a (IFN-a, 1000 U/ml) and then used for western blotting (anti- mouse RNase L, anti-HuR and anti-GAPDH). (c) Growth curve of RNASEL-knockout and wild-type MEFs. Cells were seeded in a 96-well plate with 5 Â 103 cells. Cellular growth monitoring and statistics were performed as described in Figure 1a. (d) shRNA- mediated silencing of HuR was performed with most effective shRNA clone (Supplementary Figure 3). HEK293 were transfected with shRNA-HuR or shRNA negative control for 20 h (>90% silencing; Supplementary Figure 3) followed by transfection with either RNase L expression vector or control vector for 16 h. The cells were seeded in a 96-well plate with 5 Â 103 cells and cellular growth monitoring and statistics were performed as described in Figure 1a.

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1785 Po0.001) were evident in the RNASEL- monitored for cellular growth. RNase L fails to cause knockout MEFs (Figure 2a). We have also observed antiproliferative action in HuR-silenced cells but was that in the presence of IFN there is a modest increase effective in the control (negative) shRNA-expressed cells in RNase L expression and probably an additional (Figure 2d). effect of RNase-L-mediated suppression of HuR (Figure 2b). HuR was upregulated in wild-type and RNASEL- RNase L modulation of HuR mRNA knockout MEFs both in confluent and subconfluent It was not possible to assess the mRNA half-life in MEFs due to continuous absence of RNase L (Supple- RNase-L-expressing cells due to the low levels of HuR; mentary Figure 2). The RNASEL-null MEFs had thus, we used the RNASEL-knockout MEF lines. increased growth rates compared to the wild-type cells Experiments were performed with semi-quantitative (Figure 2c). Doubling times indicated that RNASEL- PCR; the one-phase exponential decay model using null cells divide 2.7-fold faster than wild-type cells b-actin-normalized signals was used. The HuR mRNA (Po0.001). stability was enhanced in the RNASEL-null fibroblasts. To examine the impact of HuR knockdown on The half-life of HuR mRNA in wild-type fibroblasts was RNase-L-induced antiproliferative action, we first eval- 1.5 h in contrast to more than 6 h in RNASEL-knockout uated silencing experiments with several short-hairpin cells (Figures 3a and b). Figure 3a also confirmed that (shRNA) vectors against HuR or control sequence RNase L deficiency led to increased HuR mRNA levels (Supplementary Figure 3) in HEK293 cells. The most (levels at 0 h). effective shRNA against HuR, which gave approxi- We have fused two 30 untranslated regions (UTR) mately 95% knockdown at 20 h after transfection, was that contain U-rich/ARE-like sequence and ARE used (clone 4; Supplementary Figure 3). HuR-knock- (Figure 4a) to enhanced green fluorescent protein down cells were further transiently expressed with (EGFP) coding region as a reporter expression RNase L expression vector or control vector and system (Figure 4b). The half-life of the reporter GFP mRNA was measured by real-time RT–PCR in the mouse fibroblasts transfected with the different repor- ters. The half-life of GFP mRNA (T1/2 ¼ 0.5 h) ex- RNase L+/+ RNase L-/- pressed from the reporter construct with the ARE- 0 Post-AcD 01246 01246 containing HuR 3 UTR was significantly (Po0.01) HuR shorter than the half-life of the GFP mRNA (T1/2 ¼ 1h) Exp. 1 expressed from the control 30 UTR (Figure 4c). Further, β-ACTIN the half-life of GFP mRNA expressed from the ARE- bearing HuR 30 UTRs was longer by twofold in Post-AcD 01246 01246 RNASEL-null fibroblasts when compared to wild-type HuR cells (Figure 4c). Exp. 2 Further, we used reporter fluorescence readout in the β -ACTIN RNase-L-expressing polyclonal cells, because with the embryonic fibroblasts, it was difficult to visualize and 100 RNase L+/+ quantify fluorescence levels. The ARE-containing HuR 0 RNase L-/- 3 UTR itself caused reduction (threefold, Po0.001; 80 Student’s t-test) of reporter activity when compared to the control 30 UTR (Figure 4d). Though RNase L 60 caused reduction of reporter fluorescence from the construct with the control 30 UTR, due to the RNase 40 L suppression of cellular growth—to demonstrate this control levels % of normalized 20 effect we preferred not to normalize these experiments— it was far more potent toward the HuR 30 UTR and on 0 uPA ARE-containing 30 UTR (Figure 4d). RNase L was 01234567 able to potently (Bonferroni test for each of the column Time after AcD, hr pair against control was o0.001) downregulate fluores- 0 Figure 3 Effect of RNase L on HuR mRNA half-life. (a) RNase cence activity from fusion containing HuR 3 LÀ/Àand RNase L þ / þ mouse embryonic fibroblasts (MEFs) were UTRs with both ARE-like and ARE regions by at least treated with medium control (0 time point) or actinomycin D (5 mg/ threefold (Figure 4d) when compared to the control 30 ml) for various times (hr), as indicated. Semi-quantitative PCRs UTR (Po0.001). were performed with primers specific to HuR and b-actin cDNA. These data suggest that RNase L may generally act on PCR, with cycle number that allows at least semi-quantitative comparison, was used as described Materials and methods. Gels U-rich element containing sequences. A reverse tran- shown are from two independent experiments. (b) Signal intensities scription (RT)–PCR using specific primers to HuR on gels from (a) were quantified. One-phase exponential decay mRNA was performed on IP RNA obtained after anti- curves for relative mRNA half-life measurements in the two cell RNase L (data not shown); RNase L did not appear to types are shown. b-Actin signal-normalized density units are plotted as a function of time after the addition of actinomycin D. bind HuR mRNA directly and thus may require other Detail of the model is given in Materials and methods. interacting partners.

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1786 1 2 100 100

80 RNase L +/+ 80 RNase L -/- 60 60

40 40

control levels 20 20 HuR 3′UTR, NM_001419 % of normalized

11502096-2139 2278-2396 2761-2794 6057 0 0 01234567 0 1 2345 67 Time after AcD treatment Time after Actiomycin D treatment

100 3 U-rich/ARE-like GTTTATTTATACCTATTTATGTATTTATTTATTG 80 T1/2 T1/2 3'UTR RNase L+/+ RNase L-/- 60 1 1.04 1.45* 3'UTR 40 2 0.88 1.38** 1 CMV P Reporter EEF1A1 20 3 0.5 1.03*** 0 2 CMV P Reporter HuR, 1.2 kb 01234567 Time after actinomycin D treatment 3 CMV P Reporter HuR, 1.8 kb neo neo/RNase L ATCCATGGCTGTATTTATCTGTTTATTTATACCT 4 ATT TATGTATTTATTTATTGAAGTGTGAAATTG 12 fold

4 CMV P Reporter uPA 3 18 fold *** TATTTATATTTCACTATTTTTATTTATATTTTTGTAATTTTA *** 2 3'UTR Construct 24 fold ***

1 6 fold

2 4 8 16 32 64 128

% of control, log2 scale Figure 4 Response of HuR 30-UTR-mediated reporter activity to RNase L. (a) The positions of U-rich/ARE-like regions and ARE in the 30 UTR of HuR mRNA reference record. (b) Schematic diagram of reporter constructs fused with control EEF1A1 30 UTR (construct 1), HuR 30 UTR U-rich/ARE-like region (construct 2), HuR ARE-containing 30 UTR (construct 3) and urokinase plasminogen activator (uPA) 30 UTR (construct 4). The shaded boxes show recognizable ARE of HuR 30 UTR and the known uPA ARE. (c) Half-life measurements of green fluorescent protein (GFP) mRNA expressed from reporter constructs. RNase LÀ/À and RNase L þ / þ mouse embryonic fibroblasts (MEFs) were treated with medium control (0 time point) or actinomycin D (5 ug/ml) for various times, as indicated. The b-actin-normalized mRNA levels were quantified by real-time PCR using TaqMan primers/probe specific to EGFP and b-actin as described in Materials and methods. The reference point was 100% of control (time 0, RNase L þ / þ ). Half-life determinations were assessed using one-phase exponential decay model as described in Materials and methods. Data are from two independent experiments. (d) Cell lines stably expressing RNase L/neo and vector/neo in 96-well plates were transfected with the different 30-UTR constructs as indicated. Numbers in x axis refer to the constructs as outlined in (b). Readings are mean±s.e.m. of fluorescence intensities from quadruplicate wells. ***P-value o0.001 with Bonferroni post-tests and two-away analysis of variance (ANOVA).

Dependence of RNase L activity on confluency, cell cycle in RNase-L-expressing cells at the G1/S transition and nuclear/cytoplasmic distribution (Figure 5b). In contrast, when G1/S-arrested cells were The effect of RNase L on cellular growth appeared to be released by trypsinizing the cells and then subculturing greater in confluent cells when compared to subcon- them to S phase, there was no effect of RNase L on fluent cells (Figures 1a and 2c). Likewise, the effect of HuR protein (Figure 5b). In this case, HuR was nuclear. RNase L on HuR levels was greater in confluent cells Likewise, asynchronized cells or cells in phases where when compared to subconfluent cells (Figure 5a). As a HuR is predominately nuclear, the effect of RNase L result, we did not observe consistently an effect of was negligible (data not shown). These data suggest that RNase L on HuR levels with subconfluent cells. To the effect of RNase L on HuR occurs when cell determine if the effect was dependent on cell cycle, the conditions favor cytoplasmic localization of HuR. RNase-L-expressing HeLa cells were synchronized in Indeed, kinetics experiments of HuR translocation late G1 with G1/S arrest using a starvation/aphidocholin monitored by both confocal microscopy and flow treatment regimen. Flow cytometry data showed that cytometry showed that HuR cytoplasmic localization the cells were indeed 75% in G1 phase and most of the was dependent on cell-cycle phase (Supplementary HuR was cytoplasmic. HuR was dramatically reduced Figure 4).

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1787 VECTOR/NEO RNASE L/NEO

CONFLUENT

SUB CONFLUENT

R R

RNASE L RNASE L VECTO VECTO 280204.wejdan.dna..002 280204.wejdan.dna..008 HUR 180 200 G1/GO G1/GO 150 160 G2+M β-ACTIN 120 LATE G1 S 120 S 90 G2+M PHASE 80 Counts 1 2 3 4 Counts 60 Apo Apo (75%) 40 30 0 0 Confluent Sub-confluent 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A 110 *** FL2-A 100 90 Vector 80 RNase L 70 60 50 40 % of control 30 20 10 S 0 PHASE 3142 280204.wejdan.dna..012 280204.wejdan.dna..011 (80%) 100 90 Lane on Blot G1/GO 80 G2+M G2+M 60 S

40 S Counts Counts Apo Apo 20 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A FL2-A Figure 5 RNase L activity during cellular growth and confluence. (a) RNase L/neomycin or vector/neomycin stably expressing cells were grown into two different densities in six-well plates, so that confluent (100% of total well area) and subconfluent (B40%) monolayers were achieved the next day. Western blots were performed using anti-HuR and anti-b-actin. The b-actin-normalized densitometry of blots (mean±s.e.m.) from three independent experiments are shown (lower panel). ***Po0.001 using Student’s t-test. (b) Cells that stably express neomycin/vector control or RNase L/neomycin were seeded on coverslips. After 20 h, the cells were serum- starved, then treated with aphidocholin for additional 20 h to arrest cells in G1/S, that is, late G1 (upper panels). Subsequently, cells were released from the arrest to enter S phase (lower panels) by removing the drug and subculturing in complete medium with 15% serum for 8h. The fluorescently labeled secondary antibody was used to reveal anti-HuR antibody using confocal microscopy. Flow cytometry was performed by propidium iodide staining.

To gain further insights to the differential effect of Discussion RNase L on HuR during confluence or cell cycle, we have looked at nuclear/cytoplasmic distribution of RNase L has an essential function in host defense, RNase L. Because of the low expression of RNase L particularly against viruses including both DNA and in the cell line used in the previous experiments, that is, RNA viruses (Li et al., 1998; Zhou et al., 1998; Khabar HeLa, we used Huh-7 liver cell line for the localization et al., 2000). Further work showed that RNase L is studies. We found that RNase L can exist in the nucleus also involved in apoptosis and in tumor suppression or in the cytoplasm when cells are subconfluent or although without known mechanisms (Zhou et al., 1997; confluent (Figure 6a). The spatial distribution of RNase Castelli et al., 1998; Carpten et al., 2002; Xiang et al., L and HuR during confluent and subconfluent cell 2003; Urisman et al., 2006; Andersen et al., 2007; Liu conditions was also verified by western blotting using et al., 2007). In this study, we demonstrated a probable nuclear and cytoplasmic fractions (Figure 6b). mechanism whereby RNase L suppresses cellular Because RNase L is continuously devoid in RNA- growth. Briefly, we have provided evidence, using both SEL-knockout MEFs, HuR upregulation should be RNase L overexpression and RNase L knockout seen in both nuclear and cytoplasmic compartments and models, that RNase-L-mediated suppression of cellular independent on confluence. Supplemental data (Supple- growth is associated with downregulation of the RNA- mentary Figures 2 and 5) show this is the case. binding protein, HuR, mRNA and protein, and

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1788 SUB-CONFLUENT CONFLUENT Although, RNase L has been primarily recognized in the past as antiviral enzyme, the data on the effect of RNase L on the mRNA stability of HuR in this study, PKR in our previous report (Khabar et al., 2003), on MyoD by other investigators (Bisbal et al., 2000) and on ISG43 (Li et al., HUR 2000), clearly indicate a regulatory function on cellular mRNAs without virus activation. The mRNA for MyoD, a muscle differentiation transcriptional factor, which itself has been shown to be a target for RNase L (Bisbal et al., 2000), was also found to be an HuR mRNA target by a previous study (van der Giessen et al., 2003). This may indirectly support our finding of RNase L action on HuR mRNA. The HuR 30 UTR contains both ARE-like and ARE, which may render the HuR mRNA more suscep- RNASE L tible to RNase L action that is known to cleave at UU or UA dinucleotides (Wreschner et al., 1981b). The effect of RNase L on cellular mRNA is not global because many control and housekeeping mRNAs are not affected (Bisbal et al., 2000; Li et al., 2000; Khabar et al., 2003). An intriguing question is: what is the source of double- Sub-confluent Confluent stranded RNA, an activator of OAS, that leads to generation of 2-5A that binds and activates RNase L? Cyt Nuc Cyt Nuc The answer may be the cellular mRNAs themselves, which HuR assume secondary RNA structures such as hairpins that can activate the OAS/RNase L. A general review of the RNase L source and fate of putative cellular double-stranded RNA is given in Wang and Carmichael (2004). In addition, cellular mRNAs capable of activating OAS/RNase L Tubulin system have been recently described (Molinaro et al., 2006; Malathi et al., 2007). HDAC The RNase L effect appears to be associated with its cytoplasmic localization that is favored in confluent Figure 6 Nuclear/cytoplasmic distribution of RNase L. (a) Huh-7 cells. It has been shown that RNase L/OAS pathway cells, which constitutively express immunofluorescently detectable fluctuates with cell confluence (Jacobsen et al., 1983; levels of RNase L, were seeded on coverslips with two different Zhou et al., 1998) and may contribute, at least partly, to densities to allow cells to reach either subconfluent (B40%) or confluent stage the next day. Cells were stained with anti-RNase L some of the observations with confluent cells. For or anti-HuR followed by secondary antibody that was either example, levels of OAS, which is an upstream activator fluorescein isothiocyanate (FITC) conjugated (green color, HuR) of the RNase L pathway, increase at confluence or TRITC conjugated (red color, RNase L) for confocal (Jacobsen et al., 1983) and in HeLa cells, and at G /S visualization. (b) Huh-7 cells were seeded with two different 1 densities to allow cells to reach either subconfluent (B40%) or transition in Daudi cells (Kumar et al., 1994). In this confluent stage the next day. Nuclear and cytoplasmic extracts were study, we show that the requirement of confluence for subjected to western blotting using antibodies to RNase L, HuR, the described RNase L activity paralleled the spatial tubulin (cytoplasmic fraction control) or HDAC (nuclear fraction distribution and localization of HuR and RNase L control) to confirm the findings in a. The blot is one of two (RNase during cell confluence stages. Thus, it appeared that L) and three (HuR) independent experiments. both RNase L cytoplasmic localization and activity are optimum in confluent cells or specifically at G1/S dependent on cytoplasmic localization of RNase L. transition. The RNase L action on HuR mRNA is HuR stabilizes key AU-rich mRNAs involved in cellular independent on IFN activity because RNase L was growth (for example, cyclin D1 and c-myc) and effective against HuR mRNA, both in the presence and angiogenesis/metastasis, such as uPA, COX-2 and absence of IFN treatment of cell culture. VEGF. HuR is well known to upregulate mRNA HuR is a nuclear shuttling protein (Keene, 1999) that targets important for cell proliferation and subsequently primarily exists in the nucleus and moves to the to increase cellular growth (Lopez de Silanes et al., 2003; cytoplasm during cellular activation and stress (Gallouzi Dormoy-Raclet et al., 2007). Thus, we have not pursued et al., 2000; Wang et al., 2000). Previous work suggests further confirmation of the well-studied pathway of that HuR translocation to the cytoplasm is temporal HuR effect on cellular growth. Instead, we have focused where it may regulate the stability or translation of cell- on RNase L suppression of cellular growth and cycle mRNAs (Atasoy et al., 1998). Also, a recent study correlation with HuR expression. found that a target for HuR is the RNase L mRNA (Li In this report, we found that RNase L influenced HuR et al., 2007), indicating the possibility for reciprocal mRNA expression by virtue of using microarray analysis regulation of RNase-L- and HuR-mediated events. on cells stably expressing moderate amounts of RNase L. Because both HuR and RNase L localize in the nucleus

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1789 during subconfluent cell stages and appear in the RNA interference using a vector-based short-hairpin siRNA cytoplasmic compartment as cells grow to confluence, Four expression plasmids coding shRNA against HuR and a RNase L may actually be a shuttling protein. An earlier negative control shR0NA, which is a scrambled artificial report showed that RNase L can exist in both cytoplasm sequence that does not match human genes, were obtained and nucleus and this can be influenced by cell confluence from Superarrays Inc. (Frederick, MD, USA). A screen for HuR knockdown efficiency was performed using HuR-GFP (Bayard and Gabrion, 1993). Thus, our finding of construct. HEK293 cells in 96-well plates were transfected with RNase L in the nucleus, despite the notion that RNase L Lipofectamine 2000 with HuR-GFP and each of the four is a cytoplasmic protein, supports this earlier report. shRNA plasmids (each is 50 ng per microwell). Fluorescence Further details of RNase L as a shuttling protein such as levels from the transfected HuR-GFP were quantitated after presence of nuclear localization signal in RNase L will 20 or 48h and compared to levels from the cells transfected require different set of experiments. with the control negative shRNA. In situations where RNase L is mutated (Urisman et al., 2006), control of HuR expression by RNase L may also be Western blotting defective. Expression of HuR and cytoplasmic localization Cells were lysed for 30 min on ice in lysing buffer that contains have been shown to be increased in several tumors such as 10 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycho- breast, colon, gastric and ovarian tumors when compared late, 0.1% SDS and protease inhibitor cocktail (Sigma, St Louis, to normal tissues (Lopez de Silanes et al., 2003; Denkert MO, USA). Fractionation of nuclear and cytoplasm components et al., 2004; Heinonen et al., 2005; Mrena et al., 2005; was performed using a kit (Biochain) according to the manufacturer’s instructions. Equal amounts of protein samples et al Mazan-Mamczarz ., 2008). Many important mRNA were subjected to electrophoresis on 10% SDS–polyacrylamide targets for HuR such as cyclin D1, COX-2 and uPA, have gels followed by transfer to nitrocellulose membranes (Hybond defined functions in tumor maintenance and metastasis. In ECL; Amersham Biosciences, Little Chalfont, UK). Membranes healthy cells, RNase L may act as a negative feedback were hybridized with primary antibody (1:500) to HuR (Santa mechanism for HuR expression. Deregulation of this Cruz Biotechnology, Santa Cruz, CA, USA), RNase L (1:500) or pathway may lead to upregulation of HuR mRNA targets b-actin (1:1000) followed by secondary horseradish peroxidase in which their gene products are involved in tumor- (HRP)-conjugated antibody. The anti-mouse RNase L, which is promoting events. raised in rabbits against an N-terminal peptide of mouse RNase L, was generated using custom peptide and antibody synthesis service (Alpha Diagnostics, San Antonio, TX, USA). Signal detection was performed with ECL western blotting detection Materials and methods reagents (Amersham Biosciences). Protein molecular weight markers were used to verify the size of the proteins. Cell lines WISH (HeLa markers) and HeLa cell lines were obtained from Real-time continuous assessment of cellular growth American Type Culture Collection (Rockville, MD, USA) and Cellular growth of cell lines expressing RNase L or shRNA cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) against HuR was assessed using the real-time electronic supplemented with 10% fetal bovine serum (FBS) and biosensor, RT-CES system (ACEA Biosciences Inc., San antibiotics. Huh-7 was obtained from Dr Stephen Polyak Diego, CA, USA). This system is described in detail elsewhere (University of Washington, Seattle, WA, USA) and was (Solly et al., 2004; Zhu et al., 2006). Cells were seeded in a propagated in Dulbecco’s modified Eagle’s medium (DMEM) special 96-well plate and incubated in the RT-CES system þ / þ medium with 10% FBS and antibiotics. RNase L MEF incubator for 3–5 days. Data were collected every 20 min À/À lines were generated from C57BL/6 mice whereas RNase L automatically by the analyser under the control of integrated À/À MEFs were generated from RNase L mice on the C57BL/6 software. For verification, a cellular growth curve was also background as previously described (Zhou et al., 1997). obtained using colorimetric growth indicator Alamar blue Immortalized post-crisis lines (10–14 passages (Zhou et al., (BioSource International, Camarillo, CA, USA) according to 1997) were initially obtained by continuous culturing and the manufacturer’s instructions. propagated in DMEM with high glucose, supplemented with

10% FBS and antibiotics (Invitrogen). The passage numbers 0 used in this study were between 30 and 37. 3 -UTR PCR, construction and cloning of reporter plasmids Regions that contain putative U-rich element sequences that correspond to the partial 30 UTRs of HuR (NM_00419; 1146- Transient and stable transfection of polyclonal cell lines 2359nt for U-rich ARE-like stretches, and 1146–2920nt for ARE- for RNase L and HuR containing transcript; Figure 3a) and urokinase activator HeLa cells were transfected with either pcDNA3.1 vector, (NM_002658; 2161-2301nt) were obtained by RT–PCR. Briefly, pcDNA3.1 containing full-length human RNase L cDNA or total RNA was extracted by Tri Reagent from LPS-induced full-length human cDNA for HuR (kindly provided by Dr THP-1 cell line. The cDNAs were amplified by PCR in which the Christoph Moroni, Basel, Switzerland). Stable transfections were forward primer contains BamH1 site (underlined) and the reverse performed in medium without serum using Lipofectamine 2000 primer with XbaI site (underlined) as follows—the HuR short 30 (Invitrogen) for 6 h, followed by replacing the medium with UTR was amplified by forward primer: 50-CAGCAGGGATCC serum-supplemented medium. After overnight, cells were treated TAACTCGCTCATGCTTTTTTTTG-30; reverse primer: 50-CG with neomycin (G418) until clones were formed. Approximately ACCTCTAGAATTCGAGCAAAACAAAATC-30.TheHuR 50 neomycin-resistant clones were pooled as a single polyclonal 30 UTR of a longer region was amplified using the following line. We have used pooled populations of drug-selected clones to primers—forward primer: 50-CAGCAGGGATCCTAACTCGC rule out possible clonal variations. Cells were only used for the TCATGCTTTTTTTTG-30; reverse primer: 50-CGACCTCTAG first 20 passages that had no loss of overexpression as determined ACACAGCCCCTCAGTAAAAGA-30. The uPA 30 UTR for- by western blotting. ward and reverse primes are as follows: 50-CAGCAGGGATCC

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1790 CACTGTCTCAGTTTCACTTT-30 and 50-CGACCTCTAGA and resuspended in ice-cold fixation solution. Cell suspensions CATCAGAAAAATCACATTTTATTG-30. The PCR products were processed for cell-cycle determination using the propi- were purified, precipitated and cut by BamH1 and XbaI dium iodide method with flow cytometry. sequentially, and subsequently ligated into EGFP plasmid (Gene Therapy Systems Inc., San Diego, CA, USA) that is under CMV/intron A constitutive promoter. After ligation and Immunofluorescence and confocal microscopy transformation, recombinant colonies were verified by PCR Cells were seeded on coverslips, mounted on slides, overnight using a forward vector-specific primer and 30 UTR or ARE to be subconfluent (40% of total well area) or confluent reverse primer. (100%) for the following day. The slides were formaldehyde- fixed for 15 min at 37 1C in PBS (pH 7.4), followed by permeabilization of the cellular membrane with 0.05% triton Reporter transfection and activity assessment for 5 min incubation and treated with 1:50 primary antibody to Reporter constructs containing GFP-30 UTR/ARE were used 4 HuR or RNase L for 16 h at 4 1C. The cells were then washed in transient transfection at 25 ng per 2 Â 10 cell per well in 96- to remove unbound antibodies and then treated with well microplates. Transfection was performed with Lipofecta- fluorescein isothiocyanate (FITC)-labeled secondary antibody mine 2000 as described earlier. In co-transfection experiments, (1:200; 1 h at 37 1C). Cells were then washed in PBS and the the expression plasmids of RNase L, HuR and mock (pcDNA fluorescence was analysed using confocal microscopy (Leica- 3.1) were kept constant at 100 ng unless otherwise indicated. Kaki, Saudi Arabia). For colocalization studies, cells were Transfection efficiency and normalization to control was 0 treated with goat anti-HuR (1:50) and mouse anti-RNase L achieved using EGFF reporter fused with stable EEF1A1 3 (1:50; C-17; Santa Cruz Biotechnology). FITC-conjugated and UTR. The intrawell variance of any replicate groups in tetramethylrhodamine isothiocyanate (TRITC)-conjugated fluorescence is generally o6%, which does not warrant secondary antibodies were used for HuR and RNase L et al intrawell normalization of transfection (Al-Zoghaibi ., antibodies, respectively. 2007). Fluorescence was quantitated using a sensitive bottom In the Supplemental Materials, the following methods are read instrument Zenyth 3100 (Anthos Labtec, Eugendorf, described: Austria) or by using image segmentation and quantitation Microarray expression and analysis algorithm (Al-Zoghaibi et al., 2007). Data are presented as ± Semi-quantitative and real-time RT-PCR mean value s.e.m. of fluorescence intensity. Statistics and image analysis Confluence and flow cytometry analysis Cells were made either subconfluent or confluent by culturing Acknowledgements the appropriate cell number overnight. Cell quiescence of normal human fibroblasts was achieved by confluence over a We thank Dr Myriam Gorospe for critical reading of the paper. period of 3 days. Cell synchronization was achieved by first We also thank Dr A Aboussekhra for his valuable comments on arresting G0 by starvation using serum-free medium for 16 h, cell-cycle experiments. We also appreciate the staff at the flow followed by synchronization at G1/S by treating cells with cytometry facility for their technical assistance. The assistance of 5 mg/ml aphidocholin (Sigma) for 20 h. To release cells in the S Dr P Mohideen, Mr Maher Al-Saif, and Mr Fahad Al-Zoghaibi phase, the cells were washed off the inhibitor and subcultured in cloning of the 30 UTR is appreciated. We especially thank Dr in complete medium with 15% serum. Cell phase status was Hitti for his help with real-time primer/probe design. We are also monitored by flow cytometry. Cells from either subconfluent indebted to the King Faisal Specialist Hospital and Research (40% of total well area) or confluent cultures were harvested Center Subsidiary in Maryland, USA for managing and by trypsinization, washed in phosphate-buffered saline (PBS) expediting reagent and supplies purchases for this study.

References

Al-Zoghaibi F, Ashour T, Al-Ahmadi W, Abulleef H, Demirkaya O, Castelli JC, Hassel BA, Maran A, Paranjape J, Hewitt JA, Li XL et al. Khabar KS. (2007). Bioinformatics and experimental derivation (1998). The role of 20-50 oligoadenylate-activated ribonuclease L in of an efficient hybrid 30 untranslated region and use in expression apoptosis. Cell Death Differ 5: 313–320. active linear DNA with minimum poly(A) region. Gene 391: Cirino NM, Li G, Xiao W, Torrence PF, Silverman RH. (1997). 130–139. TargetingRNAdecaywith20,50 oligoadenylate-antisense in respiratory Andersen JB, Li XL, Judge CS, Zhou A, Jha BK, Shelby S et al. syncytial virus-infected cells. Proc Natl Acad Sci USA 94: 1937–1942. (2007). Role of 2-5A-dependent RNase-L in senescence and long- Denkert C, Weichert W, Pest S, Koch I, Licht D, Kobel M et al. (2004). evity. Oncogene 26: 3081–3088. Overexpression of the embryonic-lethal abnormal vision-like protein Atasoy U, Watson J, Patel D, Keene JD. (1998). ELAV protein HuA HuR in ovarian carcinoma is a prognostic factor and is associated with (HuR) can redistribute between nucleus and cytoplasm and is increased cyclooxygenase 2 expression. Cancer Res 64: 189–195. upregulated during serum stimulation and T cell activation. J Cell Diaz-Guerra M, Rivas C, Esteban M. (1997). Activation of the IFN- Sci 111: 3145–3156. inducible enzyme RNase L causes apoptosis of animal cells. Virology Bayard BA, Gabrion JB. (1993). 20,50-Oligoadenylate-dependent 236: 354–363. RNAse located in nuclei: biochemical characterization and sub- Dormoy-Raclet V, Menard I, Clair E, Kurban G, Mazroui R, Di cellular distribution of the nuclease in human and murine cells. Marco S et al. (2007). The RNA-binding protein HuR promotes cell Biochem J 296(Part 1): 155–160. migration and cell invasion by stabili. Mol Cell Biol 27: 5365–5380. Bisbal C, Silhol M, Laubenthal H, Kaluza T, Carnac G, Milligan L Frevel MA, Bakheet T, Silva AM, Hissong JG, Khabar KS, Williams et al. (2000). The 20-50 oligoadenylate/RNase L/RNase L inhibitor BR. (2003). p38Mitogen-activated protein kinase-dependent and pathway regulates both MyoD mRNA stability and muscle cell -independent signaling of mRNA stability of AU-rich element- differentiation. Mol Cell Biol 20: 4959–4969. containing transcripts. Mol Cell Biol 23: 425–436. Carpten J, Nupponen N, Isaacs S, Sood R, Robbins C, Xu J et al. Gallouzi IE, Brennan CM, Stenberg MG, Swanson MS, Eversole A, (2002). Germline mutations in the ribonuclease L gene in families Maizels N et al. (2000). HuR binding to cytoplasmic mRNA is showing linkage with HPC1. Nat Genet 30: 181–184. perturbed by heat shock. Proc Natl Acad Sci USA 97: 3073–3078.

Oncogene RNase L regulation of HuR and cellular growth W Al-Ahmadi et al 1791 Heinonen M, Bono P, Narko K, Chang SH, Lundin J, Joensuu H et al. Molinaro RJ, Jha BK, Malathi K, Varambally S, Chinnaiyan AM, (2005). Cytoplasmic HuR expression is a prognostic factor in Silverman RH. (2006). Selection and cloning of poly(rC)-binding invasive ductal breast carcinoma. Cancer Res 65: 2157–2161. protein 2 and Raf kinase inhibitor protein RNA activators of 20,50- Jacobsen H, Krause D, Friedman RM, Silverman RH. (1983). oligoadenylate synthetase from prostate cancer cells. Nucleic Acids Induction of ppp(A20p)nA-dependent RNase in murine JLS-V9R Res 34: 6684–6695. cells during growth inhibition. Proc Natl Acad Sci USA 80: Mrena J, Wiksten JP, Thiel A, Kokkola A, Pohjola L, Lundin J et al. 4954–4958. (2005). Cyclooxygenase-2 is an independent prognostic factor in Keene JD. (1999). Why is Hu where? Shuttling of early-response-gene gastric cancer and its expression is regulated by the messenger RNA messenger RNA subsets. Proc Natl Acad Sci USA 96: 5–7. stability factor HuR. Clin Cancer Res 11: 7362–7368. Khabar KS, Al-Haj L, Al-Zoghaibi F, Marie M, Dhalla M, Polyak SJ Silverman RH. (2003). Implications for RNase L in prostate cancer et al. (2004). Expressed gene clusters associated with cellular biology. Biochemistry 42: 1805–1812. sensitivity and resistance towards anti-viral and anti-proliferative Solly K, Wang X, Xu X, Strulovici B, Zheng W. (2004). Application of actions of interferon. J Mol Biol 342: 833–846. real-time cell electronic sensing (RT-CES) technology to cell-based Khabar KS, Dhalla M, Siddiqui Y, Zhou A, Al-Ahdal MN, Der SD assays. Assay Drug Dev Technol 2: 363–372. et al. (2000). Effect of deficiency of the double-stranded RNA- Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. dependent protein kinase, PKR, on antiviral resistance in the (1998). How cells respond to . Annu Rev Biochem 67: presence or absence of ribonuclease L: HSV-1 replication is 227–264. particularly sensitive to deficiency of the major IFN-mediated Subbaramaiah K, Marmao TP, Dixon DA, Dannenberg AJ. (2003). . J Interferon Cytokine Res 20: 653–659. Regulation of cyclooxygenase-2 mRNA stability by taxanes. Khabar KS, Siddiqui Y, Dhalla D, Al-Ahdal MN, al-Zoghaibi F, Evidence for involvement of p38, MAPKAPK-2 and HuR. J Biol Zhou A et al. (1999). Constitutive and IFN-induced antiviral Chem 25: 25. enzyme activities: expected and surprising consequences in gene- Tran H, Maurer F, Nagamine Y. (2003). Stabilization of urokinase devoid fibroblasts. J Interferon Cytokine Res 19: 68. Annual Meeting and urokinase receptor mRNAs by HuR is linked to its of Interferon and Cytokine Research. Liebert, New York. cytoplasmic accumulation induced by activated mitogen-activated Khabar KS, Siddiqui YM, al-Zoghaibi F, al-Haj L, Dhalla M, Zhou A protein kinase-activated protein kinase 2. Mol Cell Biol 23: et al. (2003). RNase L mediates transient control of the interferon 7177–7188. response through modulation of the double-stranded RNA-depen- Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA dent protein kinase PKR. J Biol Chem 278: 20124–20132. et al. (2006). Identification of a novel gammaretrovirus in prostate Kumar R, Choubey D, Lengyel P, Sen GC. (1988). Studies on the role tumors of patients homozygous for R462Q RNASEL variant. PLoS of the 20-50-oligoadenylate synthetase-RNase L pathway in beta Pathog 2: e25. interferon-mediated inhibition of encephalomyocarditis virus repli- van der Giessen K, Di-Marco S, Clair E, Gallouzi IE. (2003). RNAi- cation. J Virol 62: 3175–3181. mediated HuR depletion leads to the inhibition of muscle cell Kumar R, Korutla L, Zhang K. (1994). Cell cycle-dependent differentiation. J Biol Chem 278: 47119–47128. modulation of alpha-interferon-inducible gene expression and Wang Q, Carmichael GG. (2004). Effects of length and location on the activation of signaling components in Daudi cells. J Biol Chem cellular response to double-stranded RNA. Microbiol Mol Biol Rev 269: 25437–25441. 68: 432–452. Lal A, Mazan-Mamczarz K, Kawai T, Yang X, Martindale JL, Wang W, Furneaux H, Cheng H, Caldwell MC, Hutter D, Liu Y et al. Gorospe M. (2004). Concurrent versus individual binding of (2000). HuR regulates p21 mRNA stabilization by UV light. Mol HuR and AUF1 to common labile target mRNAs. EMBO J 23: Cell Biol 20: 760–769. 3092–3102. Wang W, Yang X, Cristofalo VJ, Holbrook NJ, Gorospe M. (2001). Li XL, Andersen JB, Ezelle HJ, Wilson GM, Hassel BA. (2007). Post- Loss of HuR is linked to reduced expression of proliferative genes transcriptional regulation of RNase-L expression is mediated by the during replicative senescence. Mol Cell Biol 21: 5889–5898. 30-untranslated region of its mRNA. J Biol Chem 282: 7950–7960. Wreschner DH, James TC, Silverman RH, Kerr IM. (1981a). Li XL, Blackford JA, Hassel BA. (1998). RNase L mediates the Ribosomal RNA cleavage, nuclease activation and antiviral effect of interferon through a selective reduction in 2-5A(ppp(A20p)nA) in interferon-treated cells. Nucleic Acids Res 9: viral RNA during encephalomyocarditis virus infection. J Virol 1571–1581. 72: 2752–2759. Wreschner DH, McCauley JW, Skehel JJ, Kerr IM. (1981b). Li XL, Blackford JA, Judge CS, Liu M, Xiao W, Kalvakolanu DV Interferon action—sequence specificity of the ppp(A20p)nA-depen- et al. (2000). RNase-L-dependent destabilization of interferon- dent ribonuclease. Nature 289: 414–417. induced mRNAs. A role for the 2-5A system in attenuation of the Xiang Y, Wang Z, Murakami J, Plummer S, Klein EA, Carpten JD interferon response. J Biol Chem 275: 8880–8888. et al. (2003). Effects of RNase L mutations associated with prostate Liu W, Liang SL, Liu H, Silverman R, Zhou A. (2007). Tumour cancer on apoptosis induced by 20,50-oligoadenylates. Cancer Res 63: suppressor function of RNase L in a mouse model. Eur J Cancer 43: 6795–6801. 202–209. Zhou A, Paranjape J, Brown TL, Nie H, Naik S, Dong B et al. Lopez de Silanes I, Fan J, Yang X, Zonderman AB, Potapova O, Pizer (1997). Interferon action and apoptosis are defective in mice ES et al. (2003). Role of the RNA-binding protein HuR in colon devoid of 20,50-oligoadenylate-dependent RNase L. EMBO J 16: carcinogenesis. Oncogene 22: 7146–7154. 6355–6363. Malathi K, Dong B, Gale M, Jr, Silverman RH. (2007). Small self- Zhou A, Paranjape JM, Hassel BA, Nie H, Shah S, Galinski B et al. RNA generated by RNase L amplifies antiviral innate immunity. (1998). Impact of RNase L overexpression on viral and cellular Nature 448: 816–819. growth and death. J Interferon Cytokine Res 18: 953–961. Mazan-Mamczarz K, Hagner PR, Corl S, Srikantan S, Wood WH, Zhu J, Wang X, Xu X, Abassi YA. (2006). Dynamic and label-free Becker KG et al. (2008). Post-transcriptional gene regulation by HuR monitoring of natural killer cell cytotoxic activity using electronic promotes a more tumorigenic phenotype. Oncogene 27: 6151–6163. cell sensor arrays. J Immunol Methods 309: 25–33.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene