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Originally published In Press as doi:10.1074/jbc.M607939200 on January 19, 2007

J. Biol. Chem., Vol. 282, Issue 11, 7950-7960, March 16, 2007
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Post-transcriptional Regulation of RNase-L Expression Is Mediated by the 3'-Untranslated Region of Its mRNA*

Xiao-Ling Li{ddagger}§, Jesper B. Andersen{ddagger}§, Heather J. Ezelle{ddagger}§, Gerald M. Wilson§||, and Bret A. Hassel{ddagger}§1

From the University of Maryland, Departments of {ddagger}Microbiology and Immunology and ||Biochemistry and Molecular Biology, §Marlene and Stewart Greenebaum Cancer Center, Graduate Program in Molecular Medicine, Baltimore, Maryland 21201

Received for publication, August 18, 2006 , and in revised form, January 18, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The control of mRNA stability is a potent regulatory mechanism, as small changes in mRNA half-life result in dramatic changes in the mRNA available for translation into functional protein. Such post-transcriptional regulation provides a means to rapidly alter gene expression in response to diverse stimuli, and is frequently observed in genes encoding proteins with essential cellular functions such as proliferation and stress response. One of the most extensively studied RNA decay pathways involves AU-rich elements (AREs)2 present in the 3'-untranslated region (3'-UTR) of mRNAs (1). The AUUUA sequence is a loose consensus that is found in many but not all AREs; the absence of a strict sequence motif likely reflects the importance of structural components in ARE function (2). The influence of AREs on mRNA turnover occurs through an interaction with ARE-binding proteins (AREBPs) that can function to destabilize or stabilize target mRNAs by modulating access to the decay machinery. Specifically, AREs modulate deadenylation, and decapping, two critical steps in mRNA decay (3, 4); consistent with a link between mRNA stability and translation, some AREBPs function to regulate translation (5, 6). Several AREBPs have been identified, and genetic manipulation of specific AREBPs in mice, such as HuR and tristetraprolin, resulted in dramatic and complex phenotypes that are associated with inflammation and stress response (7, 8). These phenotypes underscore the critical role of AREBPs in gene regulation in physiological and pathophysiological processes. It is estimated that 5–8% of the mRNAs in the human transcriptome contain putative ARE elements (9). The current challenge lies in determining the specific mRNA targets of AREBPs that are responsible for the phenotypes associated with ARE-mediated regulation.

RNase-L is the terminal component of an RNA decay pathway known as the 2–5A system that derives its name from the 2',5'-linked oligoadenylates (pppA(2'p5'A)n, n ≥ 2) that are required for its activation (10). A family of 2',5'-oligoadenylate synthetases are activated by double-stranded RNA to polymerize ATP into 2–5A. 2–5A, in turn, binds the latent RNase-L resulting in its dimerization and activation. Activated RNase-L cleaves single-stranded viral, ribosomal, and mRNAs with a preference for UU and UA sequences. In addition to its nuclease function, a role for RNase-L in translational regulation was recently reported (11). RNase-L activity is attenuated by the inactivation of 2–5A by cellular phosphatases and a 2'-phosphodiesterase (12), and by RLI, a protein inhibitor of RNase-L (13). The 2–5A system is an established mediator of interferon (IFN)-induced antiviral activity, and genetic approaches to manipulate RNase-L expression and activity revealed that it exerts potent antiproliferative, proapoptotic, and senescence-inducing activities independent of IFN treatment and virus infection (14–16). Consistent with a broader role for RNase-L as a natural constraint on cell proliferation, RNase-L was determined to function as a tumor suppressor by mapping of the hereditary prostate cancer-1 susceptibility allele (HPC1) to the RNASEL gene locus (17), and by the association of RNASEL mutations with the disease (18). Most recently, a novel {gamma}-retrovirus was detected at a high frequency in prostate cancer patients that were homozygous for the Arg462 -> Gln RNase-L mutation, suggesting a functional overlap between its antiviral and tumor suppressor activities (19). A few cellular mRNAs that are degraded in an RNase-L-dependent manner have been identified, but a direct substrate relationship with RNase-L, and a role in mediating RNase-L antiproliferative activities, has not been established for any candidate substrate (20–22). Microarray analysis of 2–5A-induced gene expression in prostate cancer cells (23) and human diploid fibroblasts3 identified a finite number of down-regulated genes that represent candidate RNase-L substrates, and determined that a significant number of mRNAs were up-regulated following RNase-L activation. These findings suggest that the biological activities of RNase-L involve an extensive reprogramming of one or more gene regulatory networks.

As a mediator of the cellular response to microbial and antiproliferative stress stimuli, RNase-L must be rapidly activated then efficiently attenuated, to prevent the deleterious effects of its uncontrolled activity in cells. However, apart from activation by 2–5A, little is known about the regulation of RNase-L activity. 2',5'-Oligoadenylate synthetase transcription is markedly induced by IFN, antiproliferative agents, or cell stress, providing a source of 2–5A in physiological conditions that require RNase-L activity. In contrast, RNase-L mRNA and protein are present at low basal levels in most cell types, and its enzyme activity is not associated with significant changes in RNase-L transcription (24). These findings suggested that RNase-L expression may be rapidly and transiently regulated by post-transcriptional mechanisms. Specifically, the presence of a conserved, 1.75-kb 3'-UTR in the RNase-L transcript prompted us to examine the role of RNase-L mRNA stability in the regulation of its expression. The RNase-L 3'-UTR contained multiple candidate AREs, and sequential deletion of these elements identified positive and negative regulatory regions. RNP (ribonucleoprotein) immunoprecipitation revealed that the AREBP, HuR, binds to the RNase-L mRNA in intact cells, and functions to increase its mRNA half-life and expression in a manner that is dependent on the two 3'-terminal AREs. Activation of endogenous HuR in response to stress stimuli, or during myoblast differentiation, corresponded to an increase in RNase-L expression and HuR-RNase-L mRNA interaction, suggesting that HuR regulates RNase-L in physiological conditions. In addition, the HuR-dependent regulation of RNase-L expression enhanced its antiviral activity demonstrating the capacity of this regulation to impact RNase-L function. These findings provide the first description of the post-transcriptional regulation of RNase-L expression by its 3'-UTR and HuR, and identify a novel mechanism by which the activity of the 2–5A pathway is controlled.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Transfection, and Expression Constructs—293T, HeLa, 2fTGH, and C2C12 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1x antibiotic-antimycotic (Invitrogen). Cells were maintained in a humidified atmosphere of 5% CO2, 95% balanced air at 37 °C. To induce differentiation C2C12 myoblasts cells were grown to 80–90% confluence, then transferred to Dulbecco's modified Eagle's medium containing 2% fetal calf serum and Insulin-Transferrin-Selenium-A (Invitrogen) for up to 5 days. Transfection experiments were carried out using Lipofectamine 2000 as directed by the supplier (Invitrogen). RNase-L-3'-UTR expression constructs and deletion mutants used for transfection included the previously described RNASEL cDNA (25), and are described below. The HuR expression construct was a generous gift from Myriam Gorospe (NIA, National Institutes of Health). pTRE-r betaG-3'-UTR construct was generated by sequential PCR amplification of the RNase-L 3'-UTR with primers that added flanking BglII sites, cloning of the PCR product into the pCR-blunt-Topo vector (Invitrogen), and subcloning of the BglII 3'-UTR insert downstream of the rabbit beta-globin gene in the pTRER beta-wt vector (2). The pTRE-r betaG-3'-UTR construct used in the experiments presented contained the poly(A) signals from the RNase-L 3'-UTR and the vector; however, the presence of an extra poly(A) signal did not alter beta-globin-UTR expression or mRNA stability, as a construct that contained only the vector-derived poly(A) signal gave equivalent expression (data not shown). All clones were verified by sequencing.

Cloning of the RNase-L 3'-UTR and Generation of 3'-UTR Mutants—The human RNase-L 3'-UTR was amplified from human genomic DNA using primers that flanked the stop codon and the predicted polyadenylation site: forward, 5'-GTCCCTGGCATCGTGTATTCCATA-3'; reverse, 5'-CTCTCACTACATACTAGGCCCACA-3', and cloned in to pCR-blunt-Topo. The PCR product was subcloned into the unique SpeI site in the 3'-UTR of clone ZC5 in the pcDNA3.1 vector (25) to generate the full-length RNase-L construct (ZC5+, Fig. 1). To remove the poly(A) signal from the ZC5+ construct, the plasmid was amplified using forward, 5'-CACCATGGAGAGCAGGGATCAT-3', and reverse, 5'-GAGTAAGCTTCACAAATGGGC-3', primers, and subcloned in the pcDNA3.1 vector. To remove the poly(A) signal from the pTRE-rbetaG-3'-UTR construct, the forward primer, 5'-AGATCTGGACTGATTTGCTGGAG-3', and reverse primer, 5'-AGATCTGAGTAAGCTTCACAAATGGGC-3', were used and the PCR product was subcloned into the BglII sites of the parental plasmid. The cloning junctions and 3'-UTR were verified by sequencing (University of Maryland, Baltimore Biopolymer Core). The primers used for PCR in Fig. 1B to demonstrate that the 3'-UTR is contiguous with the coding region, and that the predicted poly(A) site is used are listed according to the PCR products A–D shown in Fig. 1B. PCR products A, B, and D were: forward primer, 5'-CTACCAGAACACTGTGGGTGAT-3'; reverse primer for A, 5'-CATATGCAGCATTAGGGGTCAA-3'; reverse primer for B, 5'-GAATGAGATTCCTGGAACCCCT3'; reverse primer for D, 5'-CTCTCACTACATACTAGGCCCACA-3'. For the PCR product C: forward primer, 5'-CTGGCCCAAGATTATTCATACCTAGCACTTTATAAATTTATG-3', and reverse primer, 5'-GCACCAGAAAAACGTAAGACAG-3'.

RNase-L 3'-UTR deletion constructs were generated from a ZC5+ template by PCR with pfuHF DNA Polymerase. (Stratagene, La Jolla, CA). The forward primer for all the deletion constructs was ZC5F, 5'-CACCATGGAGAGCAGGGATCAT-3'; the reverse primers for these constructs were: {Delta}7–8, 5'-GCACCAGAAAAACGTAAGACAG-3'; {Delta}5–8, 5'-CTTCTTCAGACTCTGCCAAATG-3'; {Delta}4–8, 5'-TATGTTTTGGGCCTCATCTGGA-3'; {Delta}2–8, 5'-AGCTCACACTCTCTGAGTCTCA-3'; {Delta}1–8, 5'-CATATGCAGCATTAGGGGTCAA-3'. Internal deletions of AREs 2, 3, 7, 8, and the predicted HuR binding site were generated by PCR with ZC5+ as a template (QuikChange, Stratagene). The primers used for mutagenesis were: {Delta}ARE2, forward primer, 5'-CTGGCCCAAGATTATTCATACCTAGCACTTTATAAATTTATG-3', and reverse primer, 5'-CATAAATTTATAAAGTGCTAGGTATGAATAATCTTGGGCCAG-3'; {Delta}ARE3, forward primer 5'-CCTAGCACTTTATAATGTGGTGTTATTGGTACC-3', reverse primer, 5'-GGTACCAATAACACCACATTATAAAGTGCTAGG-3'; {Delta}ARE2&3, forward primer, 5'-CTGGCCCAAGATTATTCATGTGGTGTTATTGGTACC-3', reverse primer, 5'-GGTACCAATAACACCACATGAATAATCTTGGGCCAG-3'; {Delta}ARE7, forward primer, 5'-GTATACATTACATCTGAGTCAAAACAATCCTTTAAGGTC-3'; reverse primer, 5'-GACCTTAAAGGATTGTTTTGACTCAGATGTAATGTATAC-3'; {Delta}ARE8, forward primer, 5'-GTTGATTAGGAACAAAGGCTTAAAAAATAC-3'; reverse primer, 5'-GTATTTTTTAAGCCTTTGTTCCTAATCAAC-3'. {Delta}HuR forward primer, 5'-GTGGTGGTTGAGATGGAGCCAGTACCTTAGGTTCTTTCTG-3'; reverse primer, 5'-CAGAAAGAACCTAAGGTACTGGCTCCATCTCAACCACCAC-3'.

Analysis of RNA Expression and Half-life by Quantitative Reverse Transcriptase (RT)-PCR—Total RNA was prepared using TRIzol reagent as directed by the supplier (Invitrogen). RNA was treated with DNase I (Promega, Madison, WI) at 37 °C for 30 min, followed by the addition of 1 µl of stop solution at 65 °C for 15 min prior to analysis by Northern blot or qPCR. For qPCR analysis, 2 µg of DNase I-treated total RNA was used for reverse transcription to synthesize first strand cDNA using SuperScriptaseII RT (Invitrogen). Aliquots of the first strand cDNA were used in a standard PCR mixture (Abgene, Epsom, Surrey, UK) or in real time PCR (qPCR) using SYBR Green as directed by the manufacturer (Bio-Rad). For all primer sets, reactions were first conducted using a temperature gradient to optimize annealing conditions, and the absence of primer dimers was confirmed by melting condition analysis. RT reactions carried out in the absence of RT served as a negative control, and to detect PCR products generated from contaminating genomic DNA. PCR using plasmid templates served as positive controls. The sequences of the primers for detection of RNase-L were as follows: human coding region: forward, 5'-GCTCATTTGTACTGCGTTATGC-3', reverse, 5'-CATTTTCTCAAGGAAAAGGC-3';3'-UTR (used for the analysis of beta-globin-3'-UTR): forward, 5'-GCACTGAAGAGAGCATTTGGCAGA-3', reverse, 5'-GAGCTCCTAGACTGGGTATGGGAA-3'; rpl13a: forward 5'-CTCAAGGTCGTGCGTCTG-3', reverse, 5'-TGGCTTTCTCTTTCCTCTTCTC-3'; glyceraldehyde-3-phosphate dehydrogenase: forward, 5'-GAGTCAACGGATTTGGTCGT-3', reverse, 5'-TTGATTTTGGAGGGATCTCG-3'; beta-globin: forward, 5'-TGCATCTGTCCAGTGAGGAG-3', reverse, 5'-AGCATTTGCAGAGGACAGGT-3'.

To analyze the mRNA half-life of plasmid-encoded transcripts, cells were first transfected with the indicated plasmids, then, at 24 h post-transfection, the transfected cells were trypsinized and seeded into individual plates for treatment with 5 µg/ml actinomycin D (Sigma). In this manner, samples used to determine mRNA half-life were derived from a single transfection, thereby eliminating potential differences in expression associated with variable transfection efficiencies. Total RNA was harvested at the time points indicated in the figures. Specific mRNAs were quantified by qPCR. Each analysis represents at least two independent experiments, and within an experiment, all reactions were performed in triplicate, and the Ct values were converted to RNA concentration using a standard curve. mRNA values from each time point were normalized to the constitutively expressed ribosomal protein transcript, rpl13A. mRNA values were graphed on semi-log axes, and first order turnover rates were calculated by nonlinear regression of the percentage of mRNA remaining as a function of time after actinomycin D treatment.

Western Blotting—Cell lysates were prepared using RIPA buffer (Upstate%20Biotechnology">Upstate Biotechnology, Charlottesville, VA). The protein concentration in the lysates was determined by the Bradford microassay (Bio-Rad), and an equal amount of protein (30 µg/lane unless otherwise indicated in figure legends) was separated on 10% SDS-PAGE gels. Proteins were electrotransferred to Immobilon-P membrane (Millipore). The membranes were blocked in 5% nonfat milk TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% (v/v) Tween 20) for 1 h at room temperature, and then sequentially reacted with the primary antibody for 1 h in blocking buffer and horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Sigma). The RNase-L monoclonal antibody (provided as a generous gift from Robert Silverman, The Cleveland Clinic Foundation) was used at a 1:2000 dilution. The {alpha}-actin (Sigma), neomycin-resistance (Upstate%20Biotechnology">Upstate Biotechnology), and HuR (Santa Cruz) antibodies were used at 1:1000, 1:1000, and 1:500 dilutions, respectively. The immunoreactive complex was visualized using the Pierce SuperSignal chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) and exposure to X-Omat AR film (Eastman Kodak Co.).

RNP Immunoprecipitation—RNA-protein complexes were isolated from C2C12 myoblasts at day 3.5 of differentiation by lysing cells in PBL buffer (100 mM KCl, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40) to preserve native RNA-protein interactions. Lysate supernatants were precleared for 1 h at 4 °C using 15 µg of IgG (Sigma) and 50 µl of protein A/G-Sepharose beads (Sigma) that had been swollen in NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 0.05% Nonidet P-40) supplemented with 5% bovine serum albumin. Proteins that nonspecifically bound the protein-A/G-IgG complex were removed by centrifugation to generate the precleared lysate. For RNA immunoprecipitation, protein-A beads (100 µl) were first incubated (16 h, 4 °C) with 30 µg of IgG, goat anti-TIAR (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-HuR (Upstate%20Biotechnology">Upstate Biotechnology); beads were then washed 5 times in 1 ml of NT2 buffer before the lysate was added. Precleared cell lysate (3 mg) was then added to the protein-A/G-antibody complex and rotated for 2 h at 4°C. After washing 5 times in 1 ml of NT2 buffer, the complexes were treated sequentially with DNase I (5 min at 37 °C) then Proteinase K at 25 min at 55 °C. The RNA was extracted by phenol/chloroform and precipitated in 0.3 M sodium acetate (pH 5.5), EtOH at –20 °C for 24 h and resuspended in dH2O for analysis. The presence of specific mRNAs in the immunoprecipitated complex was determined by RT-PCR using gene-specific primers from the coding region of murine MyoD and RNase-L; the primers used for this analysis were: MyoD: forward, 5'-TACCCAAGGTGGAGATCCTG-3', reverse 5'-CATCATGCCATCAGAGCAGT-3'; murine RNase-L: forward, 5'-CAATCGAGAAGTGGCTGTGA-3', reverse, 5'-ATAGGATGCTGTGGGCAAAC-3'.

Antiviral Assay—HeLa cells were co-transfected with RNase-L expression constructs, ZC5+ and pZeoHuR or empty vector control. At 24 h post-transfection, cells were reseeded into 96-well plates, and treated with IFN{alpha} (PBL Biomedical Laboratories, New Brunswick, NJ) at the indicated concentrations for 16 h, then challenged with encephalomyocarditis virus (multiplicity of infection ~0.1) for 10 h. Cell viability was analyzed using the MTT assay (Promega, Madison, WI), and the percent protection was calculated using the formula: sample value – virus control (no IFN)/cell control (no virus) – virus control (no IFN). Each value is the mean of 8 identical wells ± S.D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RNase-L mRNA Contains a Long 3'-UTR with Putative AREs—The mature human RNase-L mRNA is 4.3 kb in size, with a coding region of just 2.2 kb and an extensive UTR of ~2.1 kb (25). Primer extension analysis identified a short, ~0.37-kb 5'-UTR (27) indicating that the remaining 1.75 kb was 3'-UTR. Predicted secondary structures and poly(A) stretches in the 3'-UTR that resulted in RT pausing and oligo(dT) binding upstream of the poly(A) tail, precluded cloning of the 3'-UTR by conventional RT-based approaches. However, analysis of the human genome sequence revealed a predicted poly(A) site, and permitted PCR amplification of the 3'-UTR from genomic DNA to generate a full-length RNase-L cDNA designated ZC5+ (Fig. 1A). RT-PCR using overlapping forward and reverse primer pairs beginning in the coding region and continuing through the 3'-UTR to downstream of the predicted poly(A) site confirmed that the 3'-UTR is contiguous with the coding region, and that the predicted poly(A) site is used (Fig. 1B; NCBI accession code 810337). Specifically, identical PCR products of the expected sizes were generated from ZC5+ and from endogenous RNase-L mRNA when primers upstream of the predicted poly(A) site were used. However, PCR using a reverse primer downstream of the predicted poly(A) site produced a product from the ZC5+ construct that contained this genomic sequence, but not from the untransfected sample that contained only endogenous RNase-L mRNA (Fig. 1B, lane D). This finding is consistent with the use of the predicted poly(A) site by endogenous RNase-L, and indicated that the ZC5+ transcript was using the vector-derived poly(A) signal. Sequence analysis of the 3'-UTR revealed eight putative AREs with canonical AUUUA sequences that may serve as determinants of RNase-L mRNA stability. The extensive nature of the RNase-L 3'-UTR, and the presence of candidate AREs suggested that it may serve an important regulatory function. Cis-acting regulatory elements are predicted to be evolutionarily conserved, therefore we compared the RNase-L 3'-UTR sequence in human and mouse. The mouse RNase-L mRNA contained a 3'-UTR of 1774 bases from the stop codon to the predicted polyadenylation site, which is slightly longer than the 1736-base human sequence. Clustal W alignment of the mouse and human 3'-UTR revealed a modest sequence similarity across the entire 3'-UTR (57% identity); however, islands of high conservation that approach the 74% identity observed in the coding region, were also detected (28). In particular, a 3'-terminal region of 305 bases exhibited a striking 71% sequence identity between mouse and human, and contained two putative ARE elements in a uridine-rich (35% U) context that characterizes functional AREs. These findings identify a conserved 3'-UTR in the RNase-L mRNA that contains ARE elements and may function in the post-transcriptional regulation of RNase-L expression.

The 3'-UTR Is a Negative Regulator of RNase-L Expression and mRNA Stability—The presence of a long 3'-UTR is frequently indicative of post-transcriptional regulation of gene expression via modulation of mRNA stability; therefore, we investigated if the RNase-L 3'-UTR modulated its expression. Transfection of 2fTGH human fibrosarcoma cells with an expression construct containing the RNase-L coding region and 200 bases of 3'-UTR (designated ZC5) resulted in a robust expression of RNase-L protein (Fig. 2A); a comparable strong expression was observed with a coding region construct, demonstrating the lack of regulatory elements in the 5'-most 200 bases of the 3'-UTR (not shown). In contrast, RNase-L expression was dramatically reduced following transfection with the full-length RNase-L ZC5+ construct that contained the 3'-UTR (Fig. 2A). This construct contained poly(A) signals from the RNase-L sequence and from the vector; however, the presence of an extra poly(A) signal did not alter RNase-L expression or mRNA stability when compared with a construct that contained only the vector-derived poly(A) signal (data not shown). The transfection efficiency was determined by Western blot analysis of the vector-encoded neomycin resistance gene (neo-r); a slightly lower level of neo-r was observed in the ZC5 transfectants indicating that ZC5 was expressed to an even greater level when normalized to neo-r expression. Endogenous RNase-L was not detectable in vector-transfected controls, indicating that the RNase-L expression was derived solely from the transfected constructs. The difference in ZC5 and ZC5+ protein expression corresponded to a pronounced difference in the levels of their respective mRNAs. Indeed, qPCR analysis of steady state RNase-L mRNA revealed a 3.3-fold increase in ZC5 as compared with ZC5+ transfectants (Fig. 2B); no RNase-L PCR product was detected in vector-transfected cells, indicating that endogenous RNase-L mRNA did not contribute to the qPCR signal in the conditions used. Primers used for qPCR analyses included the coding region and 3'-UTR sequences to ensure that the complete ZC5+ transcript was expressed. To determine whether the 3'-UTR-dependent inhibition of RNase-L expression reflected a change in RNase-L mRNA stability, 293T cells were transfected with ZC5 or ZC5+ constructs, then subcultured into multiple plates for actinomycin-D treatment and analysis of mRNA turnover by qPCR. This regimen insures equivalent transfection efficiency in all samples used to calculate mRNA half-life. This analysis revealed a 3'-UTR-dependent reduction in RNase-L stability from 74 min for ZC5, to 33.5 min for ZC5+ (Fig. 2C). To examine the activity of the 3'-UTR independent of the RNase-L coding region, and determine whether it could confer destabilizing activity to a heterologous mRNA, expression constructs encoding chimeric transcripts comprised of the stable rabbit beta-globin gene fused to the RNase-L 3'-UTR (beta-globin-3'-UTR) were generated (Fig. 3A). beta-Globin-3'-UTR and parental beta-globin plasmids were transfected into 293T cells, and mRNA was measured after actinomycin-D treatment by RT-PCR (Fig. 3B). Quantitation of the mRNA by qPCR revealed a dramatic 12-fold decrease in the half-life of beta-globin-3'-UTR as compared with the construct lacking the 3'-UTR (Fig. 3C). Thus, the RNase-L 3'-UTR can act in cis to potently modulate the stability of a heterologous mRNA. These findings identify a novel role for the 3'-UTR in regulating RNase-L expression and mRNA stability.


Figure 1
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  		FIGURE 1.
Cloning and annotation of the human RNase-L 3'-UTR. A, sequence: the coding region, and the genomic sequence that is 3' of the pA addition site are shaded; the "A" of UGA stop codon is base 2393 of NCBI accession NM_021133; the stop codon and poly(A) signal are boxed; AUUUA motifs are in bold and boxed; the predicted HuR binding site is in bold italics; reverse primers used to generate the deletion constructs are underlined in bold. B, RT-PCR analysis of the RNase-L 3'-UTR. Primer locations, PCR strategy, and predicted PCR products are shown in the diagram; the solid rectangle represents the coding region, and the thin line designates the 3'-UTR. RNA samples from untransfected and ZC5+-transfected 293T cells were used for RT-PCR analysis as depicted in the diagram, and a photograph of the ethidium bromide-stained gel is shown. Product D was generated from the ZC5+ plasmid to confirm the presence of the genomic sequence downstream from the poly(A) site in that construct. Lanes are labeled corresponding to the diagram and indicate the specific PCR product generated; X, unused lane; RT-, negative control performed in the absence of RT; M, molecular mass markers are 4.0, 3.0, 2.0, 1.5, 1.0, and 0.5 kb.

 
Positive and Negative Regulatory Elements in the RNase-L 3'-UTR—Eight putative ARE elements are distributed throughout the RNase-L 3'-UTR, with the two 3'-most AREs (Fig. 4A, numbered 7 and 8) situated in a highly conserved, U-rich context that is characteristic of functional AREs. To determine the roles of specific AREs in RNase-L regulation, and map other cis-acting elements in the 3'-UTR, deletion mutants that sequentially removed AREs were generated (Fig. 4A); the polyadenylation site from the vector was retained in the deletion mutants to prevent any influence of this element on RNase-L expression. Deletion constructs were transfected into 293T or HeLa cells, and RNase-L protein and mRNA were measured by Western blot and qPCR, respectively (Fig. 4, B and C). Identical patterns of 3'-UTR regulation were observed in 293T and HeLa cells indicating that this regulation is not cell type-specific, and that the factors that mediate this regulation may be ubiquitously expressed. As previously demonstrated, the ZC5 construct containing the coding region and just 200 bases of 3'-UTR was robustly expressed, whereas RNase-L expression from the ZC5+ construct that contains the complete 3'-UTR was markedly reduced. Removal of the two 3'-most AREs ({Delta}7–8) resulted in a dramatic decrease in RNase-L mRNA and protein below that observed with the complete UTR, indicating that this region functions as a positive regulator of RNase-L expression. Deletion of upstream AREs in constructs {Delta}5–8 and {Delta}4–8 increased RNase-L expression as compared with the {Delta}7–8 construct, suggesting that this region serves a negative regulatory function. ARE 1 appeared to be without effect, as its presence in a separate construct that lacked AREs 2–8 resulted in RNase-L expression similar to that observed in ZC5 or {Delta}1–8 that lack all candidate AREs (not shown). However, restoration of AREs 2 and 3 in the {Delta}4–8 construct reduced RNase-L expression to a level comparable with that seen with the complete 3'-UTR, implicating this region in negative regulation. To directly test the negative regulatory activity of AREs 2 and 3, and the positive regulatory activity of AREs 7 and 8, in the context of the full-length 3'-UTR, these AREs were deleted independently or in combination. Specifically, constructs in which the AUUUA motif was deleted in each single ARE mutant, and a double mutant that deleted AREs 2 and 3 as well as the 17 intervening bases, were generated. In addition, a construct that deleted a predicted binding site for the AREBP, HuR, that lies between AREs 7 and 8, was generated (Fig. 1A, and see below). Transfection of the ARE 2, 3, and double mutants into 293T cells dramatically enhanced RNase-L expression as compared with that observed with the ZC5+ construct that contains both AREs 2 and 3 (Fig. 4D). Thus, deletion of either ARE independently, or in combination, had a similar effect on RNase-L expression. In contrast, deletion of AREs 7, 8, or the predicted HuR binding site did not alter RNase-L expression compared with that observed with the ZC5+ construct (Fig. 4E), suggesting that these AREs constitute interdependent elements, all of which are required for regulatory activity. These findings implicate AREs 2 and 3 in maintaining the low levels of basal RNase-L expression that are observed in most cell types, whereas AREs 7 and 8 appeared to mediate a positive regulatory function. In light of the high sequence conservation and U-rich context of the region comprising AREs 7–8, and the paucity of information on the mechanisms by which RNase-L expression is up-regulated, we focused our studies on determining the mechanism by which this cis-element regulates RNase-L expression.


Figure 2
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  		FIGURE 2.
The 3'-UTR negatively regulates RNase-L expression and mRNA stability. A, human fibrosarcoma 2fTGH cells transfected with plasmids as indicated were analyzed for RNase-L and neomycin resistance protein expression by Western blot. A diagram of the human RNase-L expression constructs is shown below the blot. B, RNase-L mRNA was analyzed by qPCR from cells treated as in A. C, 293T cells were transfected with ZC5 or ZC5+ constructs, then treated with actinomycin-D for the times indicated, and RNase-L mRNA was quantified by qPCR and normalized to the constitutively expressed rpl13a mRNA for calculation of the mRNA half-life. Open circles are values for ZC5+, and closed circles are values for ZC5.

 


Figure 3
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  		FIGURE 3.
The RNase-L 3'-UTR destabilizes beta-globin mRNA. A, diagram of beta-globin reporter constructs used in this study; black rectangles represent exons. B, 293T cells were transfected with beta-globin or beta-globin-3'-UTR constructs that lacked or contained the RNase-L 3'-UTR, respectively, then treated with actinomycin-D for the indicated times. mRNA expression was determined by RT-PCR. C, beta-globin and beta-globin-3'-UTR mRNAs were quantified by qPCR and normalized to the constitutively expressed rpl13a mRNA for calculation of the mRNA half-life. Open circles are values for beta-globin-3'-UTR, and closed circles are values for beta-globin.

 


Figure 4
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  		FIGURE 4.
Positive and negative cis-elements in the RNase-L 3'-UTR. A, diagram of RNase-L 3'-UTR deletion mutants with the positions of candidate AREs indicated. B, RNase-L 3'-UTR deletion constructs were transfected into cells as indicated, and RNase-L expression was measured by Western blot at 24 h post-transfection. The blot shown is overexposed to detect expression of the {Delta}7–8 construct. C, steady state RNase-L mRNA expression from each of the deletion constructs in HeLa cells was quantified by qPCR, and normalized to the constitutively expressed rpl13a mRNA. D and E, RNase-L expression constructs including those with internal deletions of AREs 2, 3 (D), and 7, 8, and the predicted HuR binding site (E) were transfected into 293T cells, and expression was measured by Western blot.

 
HuR Regulates RNase-L Expression in a 3'-UTR-dependent Manner—The impact of AREs on mRNA stability occurs through their interaction with RNABPs and associated proteins. HuR is an RNA-binding protein that stabilizes ARE-containing mRNAs, and deletion analysis of the RNase-L 3'-UTR identified a strong positive regulatory element in the region containing the two 3'-most AREs (Fig. 4); therefore, we hypothesized that HuR may mediate the increase in RNase-L expression that is associated with this region of the 3'-UTR. A global screen for HuR target mRNAs by RNP immunoprecipitation (IP) recently identified a consensus sequence for HuR interaction (29), and analysis of the RNase-L 3'-UTR revealed the presence of a predicted HuR binding site located between AREs 7 and 8 (Fig. 1A). To determine the potential role of HuR in the 3'-UTR-dependent regulation of RNase-L, HeLa cells were co-transfected with ZC5, ZC5+, or {Delta}7–8 constructs, and a HuR expression construct or control vector. Analysis of RNase-L protein revealed a dramatic, 3.6-fold HuR-dependent increase in expression in ZC5+-transfected cells, whereas HuR had no effect on RNase-L expression from the ZC5 construct lacking the 3'-UTR (Fig. 5A). An identical pattern of HuR-dependent regulation was observed when 293T cells were used for transfection (not shown). Thus, HuR mediates a 3'-UTR-dependent increase in RNase-L expression. To determine whether HuR enhanced RNase-L expression through its predicted binding site in the 3' terminal region of the RNase-L 3'-UTR, HeLa cells were transfected with the {Delta}7–8 construct and HuR or vector control. HuR-dependent regulation of RNase-L expression was markedly reduced in cells transfected with construct {Delta}7–8 that lacked the predicted HuR binding site (Fig. 5A), indicating that the region deleted in construct {Delta}7–8 is essential for HuR-mediated regulation of RNase-L. However, normalization of the RNase-L signal to neo-r to account for differences in transfection efficiency revealed a 1.3-fold HuR-mediated increase in expression from the {Delta}7–8 construct suggesting that HuR may exert minor effects on RNase-L expression independent of the RNase-L 3'-UTR ARE 7–8 domain. A primary mechanism by which HuR regulates expression of its target mRNAs is through modulation of mRNA stability; therefore, we examined if the HuR-dependent increase in RNase-L protein expression reflected a change in its mRNA half-life. Co-transfection of HuR with ZC5+ resulted in a dramatic stabilization of RNase-L mRNA as compared with cells transfected with ZC5+ and control vector (Fig. 5B). In contrast, HuR had a reduced impact on the half-life of the {Delta}7–8 mRNA that lacked the predicted HuR binding site (Fig. 5C). Taken together, these findings identify a novel role for HuR in the 3'-UTR-dependent regulation of RNase-L mRNA stability and expression.

Enhanced Expression and Physical Association of RNase-L mRNA with HuR in Conditions of Endogenous HuR Activation—HuR is an established RNA-binding protein that increased the mRNA stability and protein level of ectopically expressed RNase-L in a manner that is dependent on its 3'-UTR (Fig. 5). To determine whether the endogenous RNase-L transcript is a target of HuR interaction, RNP IP was used to isolate mRNAs that interact with HuR in native conditions that preserve intracellular RNA-protein interactions (30). RT-PCR of RNA isolated from the immunoprecipitates was then used to detect the presence of RNase-L mRNA in the HuR IP complex. For this analysis, we used the C2C12 murine myoblast system in which cytoplasmic HuR protein and endogenous RNase-L mRNA are coordinately increased during differentiation (31, 32). In addition, differentiating myoblasts are one of the few cell types in that endogenous RNase-L mRNA is increased above very low basal levels (33). We first used RT-PCR analysis to confirm the induction of RNase-L mRNA during the differentiation of murine C2C12 myoblasts; the endogenous RNase-L transcript was undetectable in resting myoblasts and increased to a maximal level at day 3.5 of differentiation before returning to basal levels in fully differentiated myotubes (Fig. 6A). A concomitant increase in cytoplasmic HuR protein was observed in these samples (Fig. 6B). Accordingly, HuR was immunoprecipitated from C2C12 myoblasts at day 3.5 of differentiation, and the presence of HuR-bound RNase-L mRNA was analyzed by RT-PCR. HuR-bound RNase-L mRNA was readily detected in the IP from differentiating day 3.5 myoblasts, and corresponded to the increase in RNase-L mRNA observed at that time point (Fig. 6C). RT-PCR following immunoprecipitation with the control IgG did not result in any RNase-L-specific PCR product. To determine whether the interaction of RNase-L mRNA was specific for HuR, or if it reflected a more general interaction with AREBPs, RNP IP was performed with antibody to a distinct AREBP, TIAR (TIA-1-related). RNase-L mRNA was not detected following immunoprecipitation with the TIAR antibody indicating that the RNase-L mRNA interacts specifically with HuR (Fig. 6C); however, we cannot rule out interactions with other AREBPs not tested in this study. The Myo-D mRNA is known to interact with HuR and TIAR (29, 31), and was immunoprecipitated with both HuR and TIAR antibodies providing a positive control for the RNP IP analysis. These findings provide the first evidence of an interaction between endogenous RNase-L mRNA and the HuR IP complex in a physiological setting.


Figure 5
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  		FIGURE 5.
HuR increases RNase-L expression in a 3'-UTR-dependent manner. A, HeLa cells were transfected with RNase-L and HuR expression constructs as indicated, and RNase-L, HuR, {alpha}-actin (loading control), and neomycin resistance (transfection efficiency control) proteins were measured by Western blot and quantified by densitometry; values of the neo-r-normalized RNase-L signals are shown below the RNase-L blot. B and C, HeLa cells were co-transfected with ZC5+ or {Delta}7–8 and HuR or vector control constructs for 24 h, then RNase-L mRNA was quantified by qPCR and normalized to the constitutively expressed rpl13a mRNA at the indicated times post actinomycin-D treatment. The normalized values of mRNA remaining as a function of time are graphed, and the first order turnover rates were calculated by nonlinear regression. Open circles are values for ZC5 or {Delta}7–8 in the presence of HuR, and closed circles are values in the absence of HuR.

 


Figure 6
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  		FIGURE 6.
RNase-L mRNA physically associates with HuR in the physiological context of myoblast differentiation. A, murine C2C12 myoblasts were induced to differentiate, and RNase-L mRNA was measured by RT-PCR (A), and HuR and {alpha}-actin protein was measured by Western blot (B) on the indicated day post-induction. Lane C in A indicates negative control in which RT was omitted from the reaction. HuR and {alpha}-actin signals were measured by densitometry, and are expressed as a ratio below B. C, RNP complexes were immunoprecipitated with HuR, TIAR, or IgG antibodies as indicated. RNAs in the HuR IP complex were identified by RT-PCR with primers specific for RNase-L or MyoD.

 
It is well established that cell stress stimuli, including heat shock and UVC irradiation, result in an increase in cytoplasmic HuR, and HuR-dependent modulation of target gene expression (34, 35). To extend the analysis of RNase-L regulation to conditions of endogenous HuR activation, we examined the role of the 3'-UTR in RNase-L expression following heat shock or UVC-induced cell stress. 293T cells were transfected with ZC5 or ZC5+ for 24 h prior to treatment with heat shock or UVC radiation. Both stress stimuli induced RNase-L expression in ZC5+-transfected cells, whereas no change in expression was observed in cells transfected with the ZC5 construct that lacks the 3'-UTR (Fig. 7). These observations suggest a role for the RNase-L 3'-UTR in its stress-induced expression.


Figure 7
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  		FIGURE 7.
Cell stress increases RNase-L expression in a 3'-UTR-dependent manner. 293T cells were transfected with ZC5 or ZC5+, then heat shocked (A) or exposed to 30 joules UVC (B; times indicate recovery period) for the indicated times. RNase-L and {alpha}-actin proteins in A and B were measured by Western blot, and quantified by densitometry.

 
HuR-mediated Regulation of RNase-L Modulates Its Antiviral Activity—RNase-L is a well established mediator of cellular antiviral activity; therefore, we sought to determine whether the HuR-mediated regulation of RNase-L expression resulted in a functional impact on this biological function. HeLa cells were co-transfected with ZC5+, {Delta}7–8, or empty vector, and HuR or control vector and analyzed for antiviral activity. At 24 h post-transfection, cells were pretreated or not with IFN{alpha}, then infected with encephalomyocarditis virus (multiplicity of infection = 0.1). At 10 h post-infection, viable cells were quantified by MTT analysis. Consistent with the elevated expression of RNase-L in HuR-transfected cells (Fig. 5), enhanced antiviral activity was observed in cells co-transfected with ZC5+ and HuR as compared with cells transfected with ZC5+ alone (Fig. 8). In contrast, there was little impact of HuR in the absence of ZC5+ transfection. Interestingly, cells co-transfected with HuR and the {Delta}7–8 construct, which lacks the predicted HuR binding site, also exhibited an HuR-dependent increase in antiviral activity (Fig. 8). This finding suggested that HuR may impact RNase-L expression via sites distinct from the ARE7–8 region. Future studies with additional mutants will directly address this question.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Genes that mediate critical cellular functions in host defense, tumor suppression, and stress response, such as RNASEL, must be tightly regulated. Aberrant overexpression or activation of such genes can result in dysregulated cell death and inflammatory syndromes, whereas a deficiency in expression can result in enhanced susceptibility to microbial infections and cancer. RNase-L activity is regulated at many levels, from the expression and activation of 2',5'-oligoadenylate synthetase family members to the degradation of 2–5A by cellular enzymes. A protein inhibitor of RNase-L, RLI, has also been described (13). In contrast, there is little information on the regulation of RNASEL gene expression per se, and few examples of significant variations in RNase-L mRNA or protein from the low basal levels that are found in most cell types have been reported (24). Analysis of the RNASEL promoter failed to identify strong regulatory elements (27), suggesting that post-transcriptional mechanisms may be important in regulating RNase-L expression. In fact, ectopic expression of RNase-L results in apoptosis or senescence depending on the cell type (16), demonstrating that changes in the level of cellular RNase-L can mediate biological activities in the absence of specific inducers of its activator, 2–5A (e.g. IFN, virus infection). Accordingly, regulated, transient changes in RNase-L mRNA or protein may result in potent biological effects. Consistent with this prediction, we have characterized the extensive 3'-UTR found in RNase-L mRNA, and have determined that it functions as a positive and negative regulator of RNase-L expression and activity.


Figure 8
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  		FIGURE 8.
HuR impacts the antiviral activity of RNase-L. HeLa cells were transfected with ZC5+ or {Delta}7–8, and HuR or control vector, then pretreated with the indicated amounts of IFN{alpha} prior to infection with encephalomyocarditis virus. Murine IFN{alpha} was prepared by serial 1:3 dilutions from a 1000 unit/ml stock. Viable cells were measured by MTT at 10 h post-infection. The percent protection was calculated as described under "Experimental Procedures." Note that the standard deviation for this experiment was <0.5% of the value for each condition, therefore error bars cannot be visualized on the scale of the graph.

 
The RNase-L 3'-UTR Functions in Cis to Regulate Its Expression—Regulatory elements in the untranslated regions of mRNAs are frequently well conserved; therefore, we compared the mouse RNase-L 3'-UTR, with the human sequence to identify conserved regions that may serve a regulatory function. Islands of high sequence identity between the mouse and human orthologues were detected around several candidate AREs, consistent with the established function of these elements in post-transcriptional gene regulation. The full-length 3'-UTR exerted a potent inhibitory effect on RNase-L or beta-globin expression and mRNA stability (Fig. 2), suggesting that 3'-UTR-dependent regulation is an important mechanism by which RNase-L mRNA is maintained at low levels in the absence of cell stress. The ZC5+ construct was consistently expressed at a reduced level as compared with that of ZC5. However, differences in the exact ZC5+/ZC5 expression ratio were observed that may reflect variations in the endogenous factors that mediate this regulation. Indeed, AREBP expression and activity may be modulated by variations in cell density and proliferative status that, in turn could impact 3'-UTR mediated RNase-L expression (1). Deletion analysis revealed that the presence of AREs 2 and 3, but not 1, resulted in expression that was comparable with that of the full 3'-UTR (Fig. 4), implicating these AREs in this negative regulatory activity. Interestingly, deletion of AREs 2 and 3 independently, or in combination, resulted in a similar increase in RNase-L expression, suggesting that these AREs comprise a single regulatory element that can be functionally disrupted by the loss of either ARE (Fig. 4D). Indeed, computer modeling suggests that the ARE 2–3 region exhibits a potential secondary structure that may reflect this composite element (36).

The most highly conserved region comprised 305 bases in the 3' end of the 3'-UTR; deletion of this region reduced RNase-L expression below that observed with the full 3'-UTR, indicating that it served a positive regulatory function (Fig. 4). Independent deletion of AREs 7 and 8 that fall within this region did not mimic the reduction in RNase-L expression observed in the {Delta}7–8 mutant that lacked this entire region (Fig. 4E). Furthermore, deletion of the predicted HuR binding site that lies between AREs 7 and 8 also failed to replicate the activity of the {Delta}7–8 mutant (Fig. 4E). Thus unlike the closely spaced AREs 2 and 3, AREs 7 and 8, which are separated by 295 bases, may form interdependent structures or binding sites both of which are required for regulatory activity. Consistent with this prediction, computer modeling of the highly conserved regions around these elements, and the predicted HuR binding site, identified potential secondary structures (not shown) (36). Indeed, 3'-UTR secondary structure is an important determinant of protein interaction and corresponding regulation (1, 5). Further studies are required to analyze the formation of secondary structures in the RNase-L 3'-UTR.

Taken together, our results indicate that distinct elements in the RNase-L 3'-UTR mediate positive and negative regulatory activities. A similar bifunctional role for the 3'-UTR has been described for other labile mRNAs including p21 and cyclin D1 (37). In that study, HuR and AUF1 were both associated with p21 and cyclin D1 mRNAs in the nucleus, but independently bound these transcripts in the cytoplasm to promote stabilization or degradation depending on the physiological context. The combined impact of the multiple elements in the RNase-L 3'-UTR in distinct biological settings remains to be determined.

HuR Mediates a 3'-UTR-dependent Increase in RNase-L Expression—In light of the established role of HuR in stabilizing target mRNAs, and the paucity of information on mechanisms that enhance RNase-L expression, we focused our studies on the potential role of this positive regulatory region in RNase-L expression. HuR is a constitutively expressed AREBP that is translocated from the nucleus in resting cells to the cytoplasm in response to diverse stress stimuli (6, 38, 39). Cytoplasmic HuR is associated with an increase in stability or translation of its target mRNAs (38). Co-transfection of HuR and RNase-L 3'-UTR deletion mutants demonstrated that HuR enhanced RNase-L expression and mRNA stability in a manner dependent on the predicted HuR binding site between AREs 7 and 8 (Fig. 5). Moreover, RNase-L mRNA was identified in a HuR RNP IP complex indicating that it interacted with HuR or associated proteins (Figs. 5 and 6). As a target of HuR regulation, RNase-L may represent an important effector of biological activities associated with HuR action. Consistent with a role for RNase-L as a physiologic HuR target, activation of HuR by cell stress or myogenic differentiation correlated with an increase in endogenous RNase-L expression (Fig. 7). In addition, both HuR and RNase-L function in replicative senescence (40), cellular proliferative control (14, 41), and the innate immune response (7, 15), suggesting that HuR may regulate RNase-L in a broad spectrum of conditions. However, genetic ablation of HuR and RNase-L in specific physiological contexts is required to definitively evaluate the role of RNase-L mRNA as a HuR target. ARE-dependent gene regulation is the target of several signaling pathways, and the stabilization of ARE-containing mRNAs by HuR is frequently linked to p38 mitogen-activated protein kinase activation (42). Interestingly, pharmacologic inhibition of p38 did not alter the expression of ZC5 or ZC5+ (not shown), suggesting that the RNase-L AREs function independent of p38 signaling, or that a second stimulus, such as cytokine or microbial stimulation, is required. Finally, a novel mechanism in which HuR binding modulates the access of micro-RNAs to their target mRNAs was recently described (43). The RNase-L 3'-UTR is a predicted target for several micro-RNAs and ongoing studies are testing their roles in conjunction with HuR, in the post-transcriptional regulation of RNase-L expression.

Taken together, our findings support a model in which the RNase-L 3'-UTR functions to maintain its mRNA at low basal levels in resting cells, but acts to increase RNase-L expression in conditions of cell stress. It is likely that the changes in RNase-L mRNA resulting from alterations in its mRNA stability have not been previously detected due to their transient nature. Indeed, RNase-L up-regulation and activation provides a mechanism to rapidly change the cellular gene expression profile in response to stress stimuli; however, its expression must then be efficiently attenuated to prevent the potential deleterious effects of its widespread activity. Concurrent transcriptional changes in the expression of genes encoding RNase-L targets may function to maintain the new, stress-induced, gene expression setpoint.

3'-UTR-mediated Regulation in the Biological Activities of RNase-L—Our finding of a novel level of post-transcriptional regulation for RNase-L that was mediated by cis-elements in its 3'-UTR, and by the AREBP, HuR, suggested that this regulation may function in the established biological activities of RNase-L. In fact, ectopic HuR expression increased the antiviral activity of RNase-L (Fig. 8). In contrast, transfection of HuR alone had little impact on antiviral activity, suggesting that endogenous levels of HuR are sufficient to regulate endogenous RNase-L. Surprisingly, a HuR-dependent increase in antiviral activity was also observed in cells transfected with the {Delta}7–8 construct that lacks the predicted HuR binding site. This finding suggested that HuR may regulate RNase-L by binding to sites other than that in the ARE 7–8 region. A role for the post-transcriptional regulation of RNase-L in its antiviral and tumor suppressive functions predicts that the effectors of this regulation may be inactivated in virus-infected cells and in tumors that have escaped the constraints of RNase-L action. Consistent with this idea, HuR expression was altered in cytomegalovirus-infected cells resulting in a compromised antiviral activity (44), and aberrant regulation of HuR was detected in HPV18-associated cervical neoplasia (45). Altered expression of HuR and HuR target mRNAs has also been reported in cancers of the breast (46), gut (47), ovary (48), and colon (26). These studies suggest a role for dysregulated HuR expression in the etiologies of human viral infections and malignancies; however, the importance of RNase-L, as compared with other HuR targets, in these pathologies remains to be determined. The identification of gene products that mediate the post-transcriptional regulation of RNase-L expression, such as HuR, may reveal novel strategies to enhance RNase-L expression and activity for therapeutic applications. Interestingly, analysis of SNP data bases revealed a clustering of single nucleotide polymorphisms in the RNASEL 3'-UTR suggesting that it may be a region of genetic sequence diversity; the functional significance of these variants with respect to RNase-L expression, and the occurrence of 3'-UTR single nucleotide polymorphisms in human diseases are subjects of current investigation.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number 801337. Back

1 To whom correspondence should be addressed: 655 West Baltimore St., 9th floor BRB, Baltimore, MD 21201. Tel.: 410-328-2344; Fax: 410-328-6559; E-mail: bhassel{at}som.umaryland.edu.

2 The abbreviations used are: ARE, AU-rich element; IFN, interferon; 3'-UTR, 3'-untranslated region; 2–5A, 2',5'-oligoadenylate; AREBP, ARE-binding protein; RNP, ribonucleoprotein; IP, immunoprecipitation; TTP, tristetraprolin; HuR, Hu RNA binding protein family member; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; qPCR, quantitative PCR; RT, reverse transcriptase; TIAR, TIA-1-related. Back

3 J. B. Andersen, X. Li, and B. A. Hassel, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Myriam Gorospe, Ron Gartenhaus, and Janette Harro (University of Maryland Greenebaum Cancer Center) for critical discussion of the manuscript.



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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