Interaction of two members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with the 3′untranslated region (UTR) of the murine inducible nitric-oxide synthase (iNOS) mRNA is demonstrated in this study. An iNOS RNA-protein complex is formed using protein extracts from untreated and septic shock treated mouse liver. UV cross-linking reveals that the complex consists of at least two proteins, with apparent molecular masses of 60 and 70 kDa, respectively. The 60-kDa protein binding site lies within a 112-nt pyrimidine-rich sequence, approximately 160 nt from the coding sequence, and the RNA-protein complex can be precipitated by a monoclonal antibody directed against hnRNP I [also named polypyrimidine tract binding protein (PTB)]. The 70-kDa protein binds a 43-nt sequence near the 3′end of the 3′UTR and is immunoprecipitated by a monoclonal antibody against hnRNP L. A computer-simulated conformation of the 3′UTR suggests that both binding sites reside in regions easily accessible for a protein. Supershifts of the native RNA-protein complex could only be achieved with anti-hnRNP L, suggesting that within this multiprotein RNA complex, only hnRNP L is exposed to the antibodies, whereas the hnRNP I/PTB is mainly responsible for its interaction with the mRNA. Up-regulation of iNOS by septic shock reduces the RNA-protein complex formation, thus showing that hnRNP I/PTB and hnRNP L binding to the iNOS mRNA is modulated by inflammation. This suggests a novel function for the two previously described proteins as regulators of the iNOS gene.
The inducible nitric-oxide synthase (iNOS) is up-regulated by immunologic and inflammatory stimuli, such as lipopolysaccharides (LPS) or cytokines, resulting in increased production of nitric oxide (NO) (for review, see Rao, 2000). Induction of iNOS is part of the antimicrobial and tumoricidal actions of macrophages (Nathan and Hibbs, 1991; Cui et al., 1994) but may also have detrimental effects, such as tissue damage observed in, for example, arthritis, type I diabetes, and septic shock (Corbett and McDaniel, 1992; McCartney-Francis et al., 1993; Morikawa et al., 1999). The diversity of NO effects implies a tight regulation of its production.
Research to date has shown that regulation of the iNOS gene is complex, implicating transcriptional, post-transcriptional/translational, and post-translational mechanisms (Geller and Billiar, 1998), and several consensus sequences and corresponding trans-acting factors contributing to the transcriptional control of both the human and murine iNOSgenes have been described previously (Lowenstein et al., 1993; Xie et al., 1993; de Vera et al., 1996; Chu et al., 1998).
Compared with its transcriptional control, the post-transcriptional regulation of the iNOS gene is poorly understood yet probably an important part of the overall regulation. For example, in mouse peritoneal macrophages, transforming growth factor β1 suppresses iNOS expression by decreasing iNOS mRNA stability and translational efficiency and by increasing the degradation of iNOS protein (Vodovotz et al., 1993). On the other hand, iNOS mRNA stability has been shown to increase during induction of iNOS expression (Weisz et al., 1994). In none of the cases, however, are the mechanisms behind the events known. The 3′untranslated region (UTR) of both human and murine iNOS contains conserved AU-rich sequences, known to mediate mRNA instability in many labile cytokine and proto-oncogene transcripts (Evans et al., 1994; Ross, 1995). Whether these AU-rich sequences play a role in the iNOS mRNA stability remains unknown (Geller and Billiar, 1998). In one report, Rodriguez-Pascual et al. (2000) show the binding of the Drosophila melanogaster ELAV (embryonic lethal abnormal vision)-like protein HuR to AU-rich motifs in the 3′UTR of human iNOS mRNA and speculate that it could mediate the post-transcriptional events during the cytokine-induced expression of the human iNOS.
In the present study, we analyze the interaction of cytoplasmic liver proteins with the murine iNOS mRNA. We demonstrate that inflammation affects the interaction of a multiprotein complex formation with the 3′UTR of the iNOS mRNA. The proteins in the complex seem to be heterogeneous nuclear ribonucleoprotein I (hnRNP I) (also named polypyrimidine tract binding protein, PTB) and hnRNP L, and they bind at two distinct regions of the 3′UTR, both easily accessible fortrans-acting factors. The possible novel functions of hnRNP I/PTB and hnRNP L as regulators of the iNOS expression are discussed.
Materials and Methods
d-Galactosamine was from ICN Pharmaceuticals (Costa Mesa, CA), RNase A from Roche Diagnostics Scandinavia (Bromma, Sweden), RNase T1 from Invitrogen AB (Lidingö, Sweden), Amplitaq Gold and dNTP mix from PerkinElmer Life Sciences (Boston, MA), and T7 RNA polymerase from Promega (Madison, WI). Isotopes ([α-32P]UTP and [α-32P]dCTP) were from Amersham Biosciences (Piscataway, NJ). Protease inhibitors (phenylmethylsulfonyl fluoride, leupeptine and DTT), yeast tRNA, lipopolysaccharide (Escherichia coli, serotype 0127:B8), and homoribopolymers [poly(A), poly(U), poly(C) and poly(G)] were from Sigma-Aldrich Sweden AB (Stockholm, Sweden).
Antibodies and Oligodeoxyribonucleotides.
Monoclonal antibodies 4D11 (anti-hnRNP L) and 4F4 (anti-hnRNP C) were generously provided by Dr. Gideon Dreyfuss (Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA). Anti-hnRNP I/PTB was purchased from Zymed Laboratories (South San Francisco, CA). All of the oligodeoxyribonucleotides used in the study were from Sigma Genosys (Pampisford, Cambridgeshire, UK).
Male DBA/2J mice, 6 to 10 weeks old, were provided by Möllegaard (Ejby, Denmark). The mice were kept at the animal facility at the Biomedical Centre in Uppsala and fed chow and waterad libitum. They were allowed to acclimatize for 1 week before treatment. The mice (∼25 g) were treated intraperitoneally with 100 ng of LPS and 10 mg of d-galactosamine dissolved in saline. Control mice received saline only. After 6 h of treatment, the animals were sacrificed and livers were removed. The studies were approved by the Ethical Committee in Uppsala (Sweden) with approval number C3/1 and performed accordingly.
RNA Isolation and Northern Blot Analysis.
Total RNA was isolated from the liver samples using an RNeasy Midi kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's protocol. Total RNA (20 μg) was subjected to electrophoresis in 1.2% agarose/formaldehyde gel, transferred to a Hybond-N nylon membrane (Amersham Biosciences AB, Uppsala, Sweden) and UV cross-linked before hybridization. The 4-kilobase NotI fragment, excised from a plasmid containing the murine iNOS cDNA (kindly provided by Dr. Charles J. Lowenstein, John Hopkins University, Baltimore, MD), was radiolabeled with [α-32P]dCTP using the Megaprime labeling kit (Amersham Biosciences AB). Unincorporated nucleotides were removed by using a G-50 column (Amersham Biosciences AB). Both prehybridization and hybridization were performed at 65°C in Church buffer (Church and Gilbert, 1984) modified to contain 0.25 M NaHPO4, pH 7.2, 7% SDS, and 1 mM EDTA. Hybridization was performed overnight with 1.7 × 107 cpm of radiolabeled probe. To assess equal loading of the samples, the mRNA level of the house-keeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was measured using the radiolabeled GAPDH cDNA (BD Clontech, Palo Alto, CA).
Preparation of Liver Cytoplasmic Extracts.
For preparation of the crude cytoplasmic protein extracts, excised mouse liver was homogenized in 15 mM HEPES buffer, pH 7.6, containing 3 mM MgCl2, 40 mM KCl, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 10 ng/μl leupeptine, and 0.4% igepal. The homogenate was centrifuged at 12,000g for 10 min at 4°C. The supernatant, corresponding to the cytoplasmic extract, was aliquoted and stored at −80°C until further use. Protein concentration was measured by the method of Lowry et al. (1951).
In Vitro Transcription.
Using different sets of primers, cDNAs encoding various subfragments of murine iNOS 3′UTR and coding sequence (see Fig. 2, Table1) were obtained by PCR using the iNOS-containing plasmid as a template. All PCR reactions were performed with Taq DNA polymerase. After 10 min at 95°C followed 30 cycles: 94°C, 1 min; 52°C, 1 min; 72°C, 1 min. The nucleotide positions of the primers refer to the cDNA map as published byLowenstein et al. (1992). Primer 2 hybridizes to the pBluescript plasmid, 29 nt downstream of the NotI cloning site. All fragments produced with primer 2 were further subjected to cleavage with NotI to eliminate the plasmid sequence. Sense oligonucleotides contained 23 nt corresponding to the T7 RNA polymerase promoter. PCR-amplified products were transcribed with T7 RNA polymerase in the presence of [α-32P]UTP (800 Ci/mmol) using standard protocols (Promega). The 218- and 81-nt fragments were cut with the restriction enzymes EarI andSspI before use as templates for in vitrotranscription, to generate the resulting 112- and 41-nt probes, respectively. Unincorporated nucleotides were removed by using a G-50 column (Amersham Biosciences AB). Unlabeled RNA competitors were transcribed under similar conditions, with the radiolabeled nucleotide substituted with 0.5 mM UTP.
Binding reactions were carried out for 12 min at room temperature using 200,000 cpm of32P-labeled RNA transcript and 25 μg of protein extracts, in a total volume of 20 μl containing 10 mM HEPES, pH 7.6, with 3 mM MgCl2, 40 mM KCl, 5% glycerol, 1 mM DTT, and 1 μg of yeast tRNA. The reaction mixture was then placed on ice and exposed to UV light for 20 min in a Spectrolinker XL-1000 UV cross-linker (Spectronics, Westbury, NY). Subsequently, unbound RNA was digested with 2 μg of RNase A at 37°C for 20 min. The samples were denatured under nonreducing conditions and separated by SDS/PAGE (12%). Finally, the gel was dried and autoradiographed overnight. For competition experiments, the protein extract was preincubated for 5 or 10 min with the indicated unlabeled competitors.
The UV cross-linked complexes were immunoprecipitated essentially as described by Hamilton et al. (1993). A typical UV cross-linking was performed with 40 μg of liver cytoplasmic extract and 200,000 cpm of radiolabeled 112- or 81-nt probes, respectively. After RNase A digestion, the RNA-protein complexes were incubated with a 1:500 dilution of anti-hnRNP L (4D11), anti-hnRNP I/PTB, or anti-hnRNP C (4F4) (used as a control antibody) monoclonal antibodies for 2h at 4°C. The samples were then incubated with 30 μl of protein A-Sepharose beads (Amersham Biosciences AB) for 1 h at 4°C. The beads were recovered by brief centrifugation and washed three times in PBS. The proteins were denatured and analyzed by SDS/PAGE (12%). After drying, the gels were exposed to X-ray films.
RNA Mobility Shift Assay and Supershift Assay.
Binding conditions were the same as described above, using 5 or 25 μg of liver cytoplasmic extract, as indicated. After binding, the reaction mixtures were treated with RNase T1 (20 units) for 20 min at room temperature. The RNA-protein complexes were resolved on nondenaturing 7% polyacrylamide gels (acrylamide/bisacrylamide ratio, 37.5:1) with 50 mM Tris/glycine, pH 8.8, as running buffer. The gels were dried and visualized by autoradiography. For competition experiments, unlabeled competitors were added 10 min before the addition of the radiolabeled RNA. In supershift assays, 5 μg of protein extract was incubated with the RNA probe as described above. After RNase T1 treatment, 1 μl of anti-hnRNP L (4D11) anti-hnRNP I/PTB or anti-hnRNP C (4F4) monoclonal antibodies, respectively, were added to the mixtures and incubated for another 10 min, before loading on a native gel, as described by Min et al. (1995). In several cases, lanes of one or several gels from the same UV cross-linking or RNA mobility shift experiment with identical exposure times, have been combined into one figure.
Demonstration of High-Affinity Protein Binding to the 3′UTR of the Murine iNOS mRNA, and Dependence of the RNA-Protein Complex Formation on Septic Shock.
Mice treated with LPS andd-galactosamine develop septic shock (Barton and Jackson, 1993), a condition that induces the iNOS gene (Morikawa et al., 1999). This was used to mimic an infection and to achieve up-regulation of iNOS mRNA. As expected, an increase of iNOS mRNA was seen after 6 h of treatment (Fig.1).
Evidence suggests that iNOS induction involves post-transcriptional control of both the murine and human genes (Vodovotz et al., 1993;Weisz et al., 1994; Rodriguez-Pascual et al., 2000). To screen for possible trans-acting factors interacting with the 3′UTR of the murine iNOS mRNA, binding reactions were performed with the entire 3′UTR as a probe (see Fig. 2 and Table 1) and liver cytoplasmic protein extracts from septic shock-treated and -untreated animals. As shown in Fig. 3, at least two retarded bands could be seen in a gel mobility shift assay. Complex I (▹) shows the highest intensity with control extracts, disappearing almost entirely upon LPS-galactosamine treatment, whereas complex II (▸) has the highest intensity after the septic shock (Fig. 3). In repeated experiments, the disappearance of complex I upon LPS-galactosamine treatment was reproducible.
To determine the apparent molecular masses of the proteins interacting with the iNOS 3′UTR, we performed UV cross-linking experiments. Two complexes with apparent molecular masses of 60 and 70 kDa were resolved (Fig. 4). In accordance with the gel shift assay, the intensity of the complexes, in particular that of the 60-kDa complex, decreases by septic shock. Proteinase K treatment of the reaction mixture prevented the complex formation, demonstrating that the binding factors are proteins (data not shown).
To examine the specificity of the RNA-protein interactions, we performed competition experiments. The complex formation with iNOS 3′UTR and cytoplasmic extracts from untreated animals could be inhibited by unlabeled iNOS 3′UTR as a competitor (Fig.5A), but not by tRNA or a 660 nt fragment of the iNOS coding sequence (Fig. 5A). High concentrations of tRNA, however, reduced the complex formation. This shows a high-affinity binding of the native complex to iNOS 3′UTR. In support of this, competition experiments with UV cross-linking showed an efficient inhibition of both the 60- and 70-kDa complex formations by the unlabeled iNOS 3′UTR probe. In contrast, a much weaker inhibition was observed by the other competitors (Fig. 5b). The 70-kDa complex was more susceptible to tRNA competition than the 60-kDa complex, and was also slightly affected by the unlabeled coding sequence, reflecting differences in affinities of the two proteins to the iNOS mRNA. We also performed gel shift competition experiments with unlabeled homoribopolymers, resulting in an efficient inhibition by poly(U), and less by poly(C), poly(G), and poly(A), respectively (data not shown). This indicates that one (or several) of the proteins in the iNOS 3′UTR complex has a strong affinity for U-rich sequences.
Mapping of the Binding Regions for Proteins Involved in the 60- and 70-kDa Complexes.
The strategy for mapping the protein binding sites at the iNOS 3′UTR is shown in Fig. 2 and Table 1. A total of 11 RNA probes were prepared and used in both gel mobility shift and UV cross-linking analyses. RNA probes of 158 and 348 nt, respectively, were first created from two sections of the iNOS 3′UTR sequence, as shown in Fig. 2. We detected the gel-shifted complex, resembling the native complex, only with the longer probe, as shown in Fig.6A. Using smaller probes, the same complex was seen with the 218-nt sequence, whereas the 154-nt probe gave a higher complex also sensitive to septic shock (Fig. 6A). With the 112-nt probe, we still obtained a complex similar to that seen with the 3′UTR probe, whereas the 81-nt probe showed a profile partly similar to the 154-nt probe (Fig. 6a). It is noteworthy that with both the 348-, 218-, and 112-nt probes, the sensitivity of complex formation to septic shock, is conserved.
To search for the binding sites of the 60- and 70-kDa proteins, UV cross-linking with the relevant probes was performed. As seen in Fig.6B, both protein complexes appear with the 348-nt probe, whereas only one of the two binds the 218 and 154-nt probes, respectively (Fig. 6b). The binding site of the 60-kDa protein was located to the 112-nt probe, ∼160 nt from the coding region, and that of the 70-kDa protein to the 81-nt sequence at the 3′end of the 3′UTR, as shown in Fig. 6B. Further division of the 81-nt sequence revealed a weak complex with the 41-nt probe and a stronger one with the 43-nt sequence, possibly containing the primary binding site for the 70-kDa protein (Fig. 6B).
The primary binding sites for the two proteins were defined by using antisense oligonucleotides covering different regions of the 112- (AS1-AS6) and 81- (AS′1-AS′5) nt probes (Figs.7A and 8A). As shown in Fig. 7B, antisense oligonucleotides AS2-AS5 prevented the protein binding to the 112-nt probe. Because the region covered by these oligonucleotides is the most CU-rich in the 3′UTR, the result suggests that a polypyrimidine-rich binding protein is involved in the complex formation. For the 81-nt probe, the AS′3 prevented the RNA-protein complex formation most efficiently, followed by the AS′4 (Fig.8B). The AS′3 covers 16 nt of the 43-nt probe, which strongly binds the 70-kDa protein (Fig. 6B), and the rest of the AS′3 as well as the AS′4 cover parts of the 41-nt probe, which binds the protein less efficiently. This suggests that the binding site for the 70-kDa protein in the 3′UTR of iNOS mRNA probably is present within the sequence covered by AS′3 and AS′4, possibly involving the 16 nt at the 3′end of AS′3.
To test the affinities of the 60- and 70-kDa proteins for different nucleotides, competition assays with homoribopolymers and the 112- and 81-nt probes were performed. As shown in Fig.9A, the binding to the 112-nt probe was efficiently inhibited by the poly(U) but not by the other homoribopolymers. This, together with experiments using antisense oligoprobes, and the fact that the 112-nt sequence is extremely CU-rich (approximately 90% pyrimidines, Fig. 7A) suggest that the smaller, 60-kDa protein is a CU-rich binding protein. By contrast, none of the homoribopolymers prevented the complex formation with the 81-nt probe (Fig. 9B). Curiously, the poly(U) gave rise to a complex of higher molecular mass in addition to the 70-kDa complex. The observation that the 70 kDa protein does not interact with homoribopolymeric strands suggests that it recognizes a particular sequence at the 3′UTR (Fig.9B).
A computer analysis (Mfold program) was performed to predict the secondary structure of the entire 3′UTR sequence (Fig. 10). It seems that in the most stable conformation, the high-affinity binding sites of both proteins lie within exposed areas, easily accessible fortrans-acting factors. In addition, the tip of the loop including the binding site for the 70-kDa protein, is covered by the AS′3 oligonucleotide, which most efficiently prevented the binding in the competition assay (see Figs. 8 and10).
Identification of the 60- and 70-kDa Proteins Binding to the 3′UTR of iNOS.
Based on the characteristics of the smaller RNA-protein complex (an apparent molecular mass of 60 kDa, a high affinity for polyuridines, and a polypyrimidine-rich sequence), we postulated that this protein may be one form of the previously described hnRNP I/PTBs, 57- to 62-kDa multifunctional gene regulators (Hellen et al., 1993;Singh et al., 1995). To test this hypothesis, we performed immunoprecipitation of the UV cross-linked 112-nt RNA-protein complex. As shown in Fig. 11A, we were able to immunoprecipitate the 60-kDa complex with the anti-hnRNP I/PTB monoclonal antibody but not with the anti-hnRNP C or hnRNP L antibodies, indicating that indeed this protein could be one of the variants of the hnRNP I/PTB.
Similarly, based on the apparent molecular mass of the 70-kDa complex and on previous observations on interactions between hnRNP I/PTB and hnRNP L (Hahm et al., 1998a), we postulated that this protein could be the hnRNP L. Immunoprecipitation of the UV cross-linked 81-nt RNA-protein complex, using the hnRNP L monoclonal antibody, demonstrates that the antibody recognizes the 70-kDa protein, although the band obtained is weak (Fig. 11B). Somewhat surprisingly, with the 81-nt probe, we also obtained the 60-kDa complex when using the anti-hnRNP I/PTB antibody. However, as seen in the overexposed UV cross-linked reference sample both bands are actually present, suggesting that both proteins are able to bind the 81-nt sequence.
Gel mobility shift assays with the full-length 493 iNOS 3′UTR probe and the anti-hnRNP I/PTB or anti-hnRNP L antibodies were then performed (Fig. 12). As a negative control we used the anti-hnRNP C antibody. A supershift could be shown only with the anti-hnRNP L antibody (Fig. 12).
Several lines of evidence suggest that post-transcriptional events are important in the regulation of the iNOS expression, but only little is known of their molecular mechanisms (Vodovotz et al., 1993; Weisz et al., 1994; Rodriguez-Pascual et al., 2000). In this study, we identified two liver cytoplasmic proteins of 60 and 70 kDa, interacting with the 3′UTR of the murine iNOS mRNA, and our evidence strongly indicates that these proteins are hnRNP I/PTB and hnRNP L, respectively. Their binding to the 3′UTR of the mRNA is affected by septic shock, which suggests a role in the post-transcriptional events of iNOS induction by inflammation.
The RNA binding characteristics of the 60-kDa protein indicates that it may be one variant of the hnRNP I/PTB (Hellen et al., 1993; Singh et al, 1995). Its binding site at the iNOS mRNA is a polypyrimidine-rich sequence, showing a high nucleotide identity with known PTB consensus sequences (Singh et al., 1995): the 112-nt sequence at the iNOS 3′UTR, containing the high-affinity binding site, includes a 15-nt stretch in the 5′ end with 87% identity to the known PTB consensus sequence. In addition, a region in the middle of the 112-nt sequence show 77% identity with a 26-nt sequence in GAP-43 3′UTR mRNA, which has been reported to bind a PTB-like protein (Irwin et al., 1997). Another sequence, similar to the known hnRNP I/PTB binding sites, is found 42 nt upstream of the possible hnRNP L binding site in the iNOS 3′UTR. This may explain why the 60-kDa protein was found to interact at two distinct regions of the 3′UTR (Figs. 8 and 11B).
Similarly, the size of the other protein (70 kDa) binding to iNOS 3′UTR and the 70% nt identity of its binding site at the iNOS mRNA with a known binding site of hnRNP L in vascular endothelial growth factor 3′UTR mRNA, led us to hypothesize that this protein is the hnRNP L (Shih and Claffey, 1999).
Immunoprecipitation of the UV cross-linked complexes and supershift of the native complex confirmed that the two proteins binding to the 3′UTR of the iNOS mRNA are the hnRNP I/PTB and hnRNP L or highly similar proteins. The absence of strict consensus binding sequences in the different mRNA target molecules described in the literature suggests that the proteins have some flexibility in their RNA sequence recognition. Such flexibility has been shown for the hnRNP A1 (Burd and Dreyfuss, 1994). Alternatively, the two proteins described here could be close structural analogs of the hnRNP I/PTB and L species described previously, with modified RNA binding characteristics. This is possible, particularly in the case of hnRNP I/PTB, for which several variants have been described previously (Gil et al., 1991; Markovtsov et al., 2000).
Previous studies have shown that hnRNP I/PTB interacts with hnRNP L (Hahm et al., 1998a). Our studies are in agreement with the previous observations, although they do not reveal the exact nature of the interaction. When the major complex appearing in the gel-shift assay is UV cross-linked and resolved by SDS/PAGE, two bands of approximately 60 and 70 kDa are detected, showing that both proteins are present in the native complex (data not shown). Furthermore, the poly(U) homoribopolymer efficiently prevented the formation of the entire gel shifted complex (data not shown). Yet, of the two proteins described here, only the 60-kDa protein binding to the 3′UTR was poly(U) sensitive, thus suggesting that this protein is critical for the multiprotein complex formation. Also, the lack of native complex formation with the 81-nt probe, which contains the binding site for the hnRNP L, and where the hnRNP I/PTB seems to interact only weakly, suggests that the high-affinity binding of hnRNP I/PTB with the RNA is prerequisite for the binding of hnRNP L in the native complex. It is therefore interesting to note that only the anti-hnRNP L antibody caused a supershift of the native complex. This could indicate that whereas the hnRNP I/PTB regulates the interaction of the entire complex with the RNA, the hnRNP L is more exposed at the surface of the multiprotein complex.
So far, only one trans-acting factor has been described interacting with the 3′UTR of iNOS mRNA: Rodriguez-Pascual et al. (2000) have shown that the HuR protein interacts with AUUUA rich sequences of the iNOS mRNA in human DLD-1 cells and hypothesize that the protein may take part in mRNA stabilization. We did not find any evidence for the binding of HuR to mouse iNOS mRNA, and neither of the two proteins described here had affinity for AUUUA motifs (data not shown). An explanation for these essentially different results could be that trans-acting factors interacting with iNOS mRNA can be cell- and species-specific.
The hnRNP I/PTB seems to be involved in several processes, such as alternative splicing (Singh et al., 1995), viral replication (Chung and Kaplan, 1999), alteration of RNA conformation (Huang and Lai, 1999) and cap-independent translation through internal ribosomal entry sites (Hellen et al., 1993). The protein has been shown to shuttle between the cytoplasm and the nucleus in a transcription-linked manner (Michael et al., 1995), suggesting distinct roles in the different subcellular compartments. Irwin et al. (1997) propose that the interaction of hnRNP I/PTB with the 3′UTR of the bovine brain GAP-43 mRNA could stabilize the transcript and Kim et al. (2000b) show that the hnRNP I/PTB inhibits translation of Bip (an immunoglobulin heavy-chain binding protein) dependent on internal ribosomal entry sites.
The hnRNP L is highly homologous to hnRNP I (Piñol-Roma et al., 1989) and is also found to shuttle between the nucleus and the cytoplasm in a transcription-dependent manner (Michael et al., 1995;Hahm et al., 1998b). Several functions have been assigned to hnRNP L: e.g., modulation of the human vascular endothelial growth factor labile mRNA stability under hypoxic conditions (Shih and Claffey, 1999) and the translation of hepatitis C virus mRNA (Hahm et al., 1998b).
Both hnRNP I/PTB and hnRNP L contain four loosely conserved RNA recognition motifs, and it has been shown that they may form homodimers and/or heterodimers with other hnRNPs [e.g., the hnRNP E2, I, K and L (Kim et al., 2000a)]. It has therefore been suggested that some of their effects are exerted in concert (Hahm et al., 1998a). Potential roles of the heterodimer on RNA processing, mRNA nucleocytoplasmic transport and/or translation of some mRNAs have been proposed. It is also possible that the hnRNP I/PTB-hnRNP L complex has an RNA-binding specificity different from the individual proteins and thus exert a different function compared with the individual proteins (Hahm et al., 1998a).
In conclusion, we describe for the first time the interaction of hnRNP I/PTB and hnRNP L with the murine iNOS mRNA and the modulation of this interaction by inflammation, a strong inducer of the iNOS expression. The modulated interaction with the iNOS mRNA suggests a novel function for hnRNP I/PTB and hnRNP L. We do not know how septic shock affects the affinity of the proteins for the iNOS mRNA, but, based on their previously described functions, we can speculate on their possible roles in the iNOS induction. The hnRNP I/PTB could inhibit translation during resting conditions alone, as shown in the case of the Bip mRNA (Kim et al., 2000b), or concertedly with hnRNP L (Kim et al., 2000a). Alternatively, the two proteins may participate in the rapid iNOS mRNA degradation under noninduced conditions. Indeed, we detect a strong RNA-protein complex in resting cells where the iNOS transcripts are hardly detectable, even though significant transcription of theiNOS gene is going on (de Vera et al., 1996; Linn et al. 1997; Rodriguez-Pascual, 2000). The discrepancy between the high transcription level and low amounts of iNOS mRNA in resting cells can be explained by an efficient degradation of the iNOS transcript (Rodriguez-Pascual et al., 2000). In inflammation, this could be reversed, resulting in stimulation of gene expression (at least in part) via an increase in mRNA stability. How the inflammation-modulated binding of hnRNP I/PTB and hnRNP L to the iNOS mRNA relates to the mRNA stability and other post-transcriptional events is a focal point of our future research.
We are grateful to Dr. Charles J. Lowenstein for providing the murine iNOS cDNA plasmid and to Dr. Gideon Dreyfuss for supplying the anti-hnRNP C and anti-hnRNP L antibodies. We also thank Kyle Christian for careful reading of the manuscript.
- Received February 22, 2002.
- Accepted April 30, 2002.
- inducible nitric oxide synthase
- nitric oxide
- untranslated region
- heterogeneous nuclear ribonucleoprotein
- polypyrimidine tract binding protein
- polymerase chain reaction
- polyacrylamide gel electrophoresis
- coding sequence
- The American Society for Pharmacology and Experimental Therapeutics