The UV-inducible RNA-binding Protein A18 (A18 hnRNP) Plays a Protective Role in the Genotoxic Stress Response
2001; Elsevier BV; Volume: 276; Issue: 50 Linguagem: Inglês
10.1074/jbc.m105396200
ISSN1083-351X
AutoresChonglin Yang, France Carrier,
Tópico(s)RNA regulation and disease
ResumoWe have previously shown that specific RNA-binding proteins (RBP) are activated by genotoxic stress. The role and function of these stress-activated RBP are, however, poorly understood. The data presented here indicate that the RBP A18 heterogeneous ribonucleoprotein (hnRNP) is induced and translocated from the nuclei to the cytoplasm after exposure to UV radiation. Using a new in vitro system we identified potential cellular targets for A18 hnRNP. Forty-six mRNA transcripts were identified, most of which are stress- or UV-responsive genes. Two important stress-responsive transcripts, the replication protein A (RPA2) and thioredoxin, were studied in more detail. Northwestern analyses indicate that A18 hnRNP binds specifically to the 3′-untranslated region of RPA2 transcript independently of its poly(A) tail, whereas the poly(A) tail of thioredoxin mRNA reinforces binding. Overexpression of A18 hnRNP increases the mRNAs stability and consequently enhances translation in a dose-dependent manner. Moreover, cell lines expressing reduced levels of A18 hnRNP are more sensitive to UV radiation. These data suggest that A18 hnRNP plays a protective role against genotoxic stresses by translocating to the cytosol and stabilizing specific transcripts involved in cell survival. We have previously shown that specific RNA-binding proteins (RBP) are activated by genotoxic stress. The role and function of these stress-activated RBP are, however, poorly understood. The data presented here indicate that the RBP A18 heterogeneous ribonucleoprotein (hnRNP) is induced and translocated from the nuclei to the cytoplasm after exposure to UV radiation. Using a new in vitro system we identified potential cellular targets for A18 hnRNP. Forty-six mRNA transcripts were identified, most of which are stress- or UV-responsive genes. Two important stress-responsive transcripts, the replication protein A (RPA2) and thioredoxin, were studied in more detail. Northwestern analyses indicate that A18 hnRNP binds specifically to the 3′-untranslated region of RPA2 transcript independently of its poly(A) tail, whereas the poly(A) tail of thioredoxin mRNA reinforces binding. Overexpression of A18 hnRNP increases the mRNAs stability and consequently enhances translation in a dose-dependent manner. Moreover, cell lines expressing reduced levels of A18 hnRNP are more sensitive to UV radiation. These data suggest that A18 hnRNP plays a protective role against genotoxic stresses by translocating to the cytosol and stabilizing specific transcripts involved in cell survival. RNA-binding protein ribonucleoprotein heterogeneous RNP untranslated region chloramphenicol acetyltransferase green fluorescent protein replication protein A open reading frame polymerase chain reaction bovine serum albumin phosphate-buffered saline RNA binding buffer trans-activation-responsive element The cellular response to genotoxic and non-genotoxic stresses is complex. It includes multiple regulatory mechanisms that are generally thought to have protective roles. Cells respond to stress in a limited number of ways by adjusting regulatory components of basic processes such as replication, transcription, and/or translation. Much emphasis has been put on stress responses involving replication or the activation of specific genes in response to DNA damage (1Fornace Jr., A.J. Jackman J. Hollander M.C. Hoffman-Liebermann B. Liebermann D.A. Ann. N. Y. Acad. Sci. 1992; 663: 139-153Crossref PubMed Scopus (174) Google Scholar); however, the regulation of post-transcriptional and translational events in response to stress has not been studied extensively. Post-transcriptional regulation can be mediated through interaction of regulatory proteins with an mRNA 3′ end (2Gray N.K. Wickens M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399-458Crossref PubMed Scopus (449) Google Scholar). This mechanism, which occurs in several organisms, is not fully understood. Most regulations of this type have been observed during early development of different organisms from Caenorhabditis elegans to mammals (3Huarte J. Stutz A. O'Connell M.L. Gubler P. Belin D. Darrow A.L. Strickland S. Vassalli J.D. Cell. 1992; 69: 1021-1030Abstract Full Text PDF PubMed Scopus (204) Google Scholar). One possible mechanism by which regulation of translation initiation can be mediated through the 3′ end of an mRNA transcript has suggested that specific proteins bound to this region could contact the basal translation apparatus and influence translational activation or repression (2Gray N.K. Wickens M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399-458Crossref PubMed Scopus (449) Google Scholar). A recent review (4Shyu A.B. Wilkinson M.F. Cell. 2000; 102: 135-138Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) described the possibility for RNA-binding proteins to shuttle between cellular compartments either constitutively or in response to stress and regulate the localization, translation, or turnover of mRNAs. Post-transcriptional regulation can also occur through mRNAs stabilization. Recent studies describe the stabilization of mRNAs by specific RBPs1 in response to hypoxia (5Paulding W.R. Czyzyk-Krzeska M.F. J. Biol. Chem. 1999; 274: 2532-2538Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) or extracellular signals (6Westmark C.J. Malter J.S. J. Biol. Chem. 2001; 276: 1119-1126Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In a previous study (7Carrier F. Gatignol A. Hollander M.C. Jeang K.T. Fornace Jr., A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1554-1558Crossref PubMed Scopus (25) Google Scholar) we have shown that RNA binding activity of specific proteins can be induced by DNA-damaging agents. Induction of an RBP at the mRNA levels was first reported for the A18 hnRNP after UV radiation (8Fornace Jr., A.J. Alamo Jr., I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). The hnRNPs are a sub-group of ribonucleoproteins (RNPs) found in the nucleus and involved in RNA processing (9Soulard M. Della Valle V. Siomi M.C. Pinol-Roma S. Codogno P. Bauvy C. Bellini M. Lacroix J.C. Monod G. Dreyfuss G. Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar). The A18 hnRNP was originally cloned by hybridization subtraction on the basis of rapid induction in UV-irradiated Chinese hamster ovary cells (8Fornace Jr., A.J. Alamo Jr., I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). Since then, the human A18 hnRNP was cloned and characterized (10Sheikh M.S. Carrier F. Papathanasiou M.A. Hollander M.C. Zhan Q., Yu, K. Fornace Jr., A.J. J. Biol. Chem. 1997; 272: 26720-26726Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The human A18 hnRNP is a rather unique RNP. In addition to containing a conserved RNA binding domain, it also contains several repeats of an RGG box in its auxiliary domain (10Sheikh M.S. Carrier F. Papathanasiou M.A. Hollander M.C. Zhan Q., Yu, K. Fornace Jr., A.J. J. Biol. Chem. 1997; 272: 26720-26726Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The RGG boxes were first identified as single-stranded nucleic acid binding motifs in an hnRNP that does not contain the conserved RNA binding domain (11Kiledjian M. Dreyfuss G. EMBO J. 1992; 11: 2655-2664Crossref PubMed Scopus (517) Google Scholar). The auxiliary domains of RNPs are associated with protein-protein interaction (9Soulard M. Della Valle V. Siomi M.C. Pinol-Roma S. Codogno P. Bauvy C. Bellini M. Lacroix J.C. Monod G. Dreyfuss G. Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar); therefore, the presence of single-stranded nucleotide binding domains in this location is unusual. In this report we show that the nuclear A18 hnRNP is not only induced but also translocated to the cytoplasm in response to UV radiation. In addition, we established an in vitro system to isolate and identify A18 hnRNP most probable mRNA targets. Forty-six mRNAs transcripts have been identified, a large proportion of which are UV- or stress-responsive. Our data indicate that A18 hnRNP binds specifically to the 3′-UTR of the replication protein A (RPA2) and the human thioredoxin mRNAs. Co-transfection of A18 hnRNP with a CAT expression vector resulted in increased message stability and CAT activity. Moreover, cells expressing reduced levels of A18 hnRNP are more sensitive to UV radiation. Taken together these data suggest that stabilization of stress-responsive transcripts by A18 hnRNP may protect the cells against genotoxic insults. To express A18 hnRNP in mammalian cells, the open reading frame (ORF) of A18 hnRNP cDNA was cloned into theHindIII/XhoI sites of pcDNA3.1 (Invitrogen, Carlsbad, CA). The A18 hnRNP-GFP fusion expression vector was constructed by amplifying the coding region of GFP from pEGFP (Invitrogen) and cloning it downstream of A18 hnRNP into pcDNA3.1 (Invitrogen). For constitutive expression of the CAT protein, the ORF of CAT was cloned into the NheI/BamHI sites of pcDNA3.1 (Invitrogen) to generate the plasmid pcDNA3.1-CAT (CAT). The 3′-UTR of RPA2 was amplified by PCR from the full-length cDNA of RPA2 and cloned into the BamHI/XhoI sites of pcDNA3.1-CAT to generate the plasmid pcDNA3.1-CAT/RPA2–3′-UTR (CAT-UTR). To generate the antisense A18 hnRNP vector, the ORF of A18 hnRNP was cloned in antisense orientation into the HindIII/XhoI sites of pcDNA3.1. The human colorectal carcinoma RKO cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. For transient transfection, cells were cultured to 80% confluency, and the indicated plasmids were transfected with Fugene 6 lipid mix (Roche Molecular Biochemicals). A total amount of 11 μg of plasmids DNA was maintained by supplementing with pcDNA3.1. Each dish received 1 μg of CAT or CAT-UTR expression vector and up to 10 μg of A18 hnRNP. Forty-eight hours after transfection, cells were harvested and lysed for further analyses. Analyses were performed in triplicate. For stable transfection, hnRNP A18-GFP was transfected into 50% confluent RKO cells. Selection was performed with 400 μg/ml hygromycin B for 2 weeks. Clonogenic survival after UV irradiation was performed by colony formation assay. The RKO antisense cell line was established by stable transfection of the pCDNA3.1 expression vector containing the A18 hnRNP ORF in the antisense orientation into RKO cells. Single colonies were expanded, and expression levels of A18 hnRNP were verified by Western blot. About 200 cells were seeded/100-mm dish for overnight growth, then the medium was removed, and the cells were irradiated with the indicated dose of UV. Treatments were performed in triplicate for each dose. Two weeks later, colonies containing more than 50 cells were counted. The CAT activity was measured essentially as described before (12Carrier F. McCary J.M. Bae I. Yarosh D.B. Fornace Jr., A.J. AIDS Res. Hum. Retroviruses. 1994; 10: 767-773Crossref PubMed Scopus (2) Google Scholar). Cellular extracts containing either 50 or 25 μg of protein were incubated at 37 °C for 1–3 h and separated on TLC plates. The conversion of chloramphenicol was quantified on a PhosphorImager (Molecular Dynamics STORM) with the ImageQuant software. The A18 hnRNP-GFP-overexpressing cells were grown on coverslips and treated with UV radiation at a dose of 20 J/m2. Cells were put back into culture for 3 h, and fluorescence was observed with a fluorescence microscope (Zeiss, Axioskop, objective 10×, HB100-W mercury lamp). The hnRNP A18 cDNA-coding region (516 base pairs) (10Sheikh M.S. Carrier F. Papathanasiou M.A. Hollander M.C. Zhan Q., Yu, K. Fornace Jr., A.J. J. Biol. Chem. 1997; 272: 26720-26726Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) was amplified by PCR and cloned into the NdeI and XhoI sites of pET21a (Novagen, Madison, WI). Expression was achieved inEscherichia coli BL21(DE3) as recommended by the manufacturer (Novagen) except that the bacteria were grown at 30 °C. Soluble proteins were loaded on a nickel nitrilotriacetic acid column (Novagen) and stepwise eluted with 100–350 mm imidazole. The imidazole was removed by gel filtration on a D-Salt Excellulose desalting column (Pierce). The recombinant protein was finally dialyzed against 50 mm sodium phosphate buffer, pH 7.5. pT7TAR was transcribed and labeled as described before (7Carrier F. Gatignol A. Hollander M.C. Jeang K.T. Fornace Jr., A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1554-1558Crossref PubMed Scopus (25) Google Scholar). Different regions of RPA2 and thioredoxin cDNAs were amplified by PCR with a T7 promoter (5′-AGTGAATTGTAATACGACTCACTATAGGGC-3′) engineered at their 5′ ends and in vitro transcribed with MAXI-scripts (Ambion Inc., Austin, TX). The probes for RPA2 were 5′-UTR (−300 to −1), ORF (+1 to +813), 3′-UTR (+814 to +1427), and 3′-UTR + poly(A) (30Gray N.K. Coller J.M. Dickson K.S. Wickens M. EMBO J. 2000; 19: 4723-4733Crossref PubMed Scopus (195) Google Scholar). The probes for thioredoxin were 5′-UTR + ORF (−98 to +318), ORF (+1 to +318), 3′-UTR + ORF + poly(A) (30Gray N.K. Coller J.M. Dickson K.S. Wickens M. EMBO J. 2000; 19: 4723-4733Crossref PubMed Scopus (195) Google Scholar), 3′-UTR (+319 to +455), and 3′-UTR + poly(A) (30Gray N.K. Coller J.M. Dickson K.S. Wickens M. EMBO J. 2000; 19: 4723-4733Crossref PubMed Scopus (195) Google Scholar), full-length (−98 to +485). All DNA fragments were gel-purified and in vitro transcribed into RNA as described above and used for Northwestern blot analyses. Recombinant A18 hnRNP and nucleolin were coated in Nunc-Immuno tubes (immunotubes) (Nalgen Nunc International, Rochester, NY) at a concentration of 40 μg/ml in carbonate buffer, pH 9.6, overnight at 4 °C. As a negative control, bovine serum albumin (BSA) with the same concentration or buffer alone was used. The tubes were washed twice with PBS containing 0.1% Tween 20 and washed two more times with PBS. Nonspecific sites were blocked with 2% milk in PBS containing 2 μg/ml yeast tRNA for 1 h at room temperature. The tubes were washed 3 times with PBS and 3 times with RNA binding buffer (RBB, 20 mm Tris-HCl, pH 7.5, 60 mm KCl, 1 mm MgCl2, 0.2 mm EDTA, 10% glycerol). Labeled TAR RNA (106 cpm) was incubated in the immunotubes with 1 ml of RBB containing 100 units of RNase inhibitor and 2 μg of yeast tRNA. After incubation for 2 h at room temperature, the tubes were washed 10 times with 1 ml of RBB and 5 times with 1 ml of PBS. Bound RNA was then eluted stepwise with 1 ml of RBB containing either 500 mm, 1 m, or 2m NaCl. Aliquots (100 μl) of each elution fraction were counted, and binding specificity was obtained by subtracting the BSA-coated tubes counts from the A18 hnRNP-coated tubes counts. Human colon carcinoma (RKO) cells were irradiated with UV at a dose of 20 J/m2. Two hours later the cells were harvested, and the mRNA was prepared by acid phenol extraction as described previously (13Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Ten μg of mRNA were incubated in the immunotube coated with A18 hnRNP at room temperature and mixed gently by inversion for 2 h. The tube was then washed 10 times with 1 ml of RBB and 5 times with 1 ml of PBS to remove the unbound mRNA. After the last wash, the bound mRNA was eluted stepwise with 1 ml of RBB containing either 500 mm, 1 m, or 2 m NaCl. The eluted mRNA was precipitated with ethanol and used for cDNA synthesis. The first strand cDNA was synthesized using the SmartII cDNA synthesis oligo (CLONTECH, Palo Alto, CA) and superscript reverse transcriptase (Life Technologies, Inc.). The reaction was performed in 20 μl at 42 °C for 1 h. Five μl of the reaction mixture was used to amplify the double strand cDNA by long distance PCR using SmartII cDNA PCR amplification oligo (CLONTECH). PCR was performed for 30 cycles at 95 °C for 1 min, 65 °C for 1 min, and 68 °C for 6 min. The amplified cDNA was purified, ligated to pGEM-T vector (Promega, Madison, WI), and transformed into E. coli DH5α. Positives colonies were picked for further analysis by PCR fingerprinting and sequencing. Sequencing was performed on an Applied Biosystems 373 (ABI, Foster City, CA) automated sequencer. Northwestern blots were performed essentially as previously described (7Carrier F. Gatignol A. Hollander M.C. Jeang K.T. Fornace Jr., A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1554-1558Crossref PubMed Scopus (25) Google Scholar). RNase protection was performed using the ribonuclease protection kit RPAII (Ambion). Briefly, total RNA was prepared from the cells co-transfected with A18 hnRNP and the CAT reporter gene constructs. A 100-base pair fragment upstream of the stop codon of the CAT open reading frame was in vitro transcribed into the complement RNA probe and purified. For each reaction, 20 μg of total RNA was co-precipitated with the RNA probe (2 × 104 cpm for 1 h) and hybridized overnight at 42 °C. For control, 20 μg of yeast RNA was co-precipitated with the RNA probes. The hybridized RNA probe was digested with RNase A/T and precipitated. Protected RNA was separated on a 6% polyacrylamide gel. The A18 hnRNP was originally cloned by hybridization subtraction on the basis of rapid induction after UV radiation (8Fornace Jr., A.J. Alamo Jr., I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). Since then, the human gene has been cloned and shown also to be UV-inducible (10Sheikh M.S. Carrier F. Papathanasiou M.A. Hollander M.C. Zhan Q., Yu, K. Fornace Jr., A.J. J. Biol. Chem. 1997; 272: 26720-26726Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Because some hnRNPs are known to shuttle between cell compartments either constitutively (14Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (717) Google Scholar) or in response to stress (15van der Houven van Oordt W. Diaz-Meco M.T. Lozano J. Krainer A.R. Moscat J. Caceres J.F. J. Cell Biol. 2000; 149: 307-316Crossref PubMed Scopus (285) Google Scholar), we evaluated the cellular location of A18 hnRNP after UV radiation. To follow A18 hnRNP protein in the RKO cells, we fused the A18 hnRNP protein to GFP and established a stable cell line with the recombinant expression vector. Our data (Fig. 1) indicate that most cells have incorporated the fused protein and distinctly show that A18 hnRNP is a nuclear protein (panel B). Overexpression of A18 hnRNP in the absence of stress does not affect cellular growth or induce apoptosis (data not shown). Three hours after exposure to UV radiation (20 J/m2), the protein becomes clearly visible in the cytoplasm of many cells (panel D). Cytoplasmic mRNAs are thus likely to be targeted by the RNA-binding protein A18 hnRNP after UV exposure. To identify A18 hnRNP potential mRNAs targets, we developed an affinity binding technique with immunotubes. We first optimized the RNA binding conditions for A18 hnRNP in immunotubes by using a labeled TAR RNA probe. This probe was selected based on its high affinity for stress-activated RNA-binding proteins (7Carrier F. Gatignol A. Hollander M.C. Jeang K.T. Fornace Jr., A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1554-1558Crossref PubMed Scopus (25) Google Scholar). To evaluate the extent of nonspecific binding, we treated four tubes in parallel. For controls, one tube was left uncoated to estimate the nonspecific binding of RNA to the tube walls, and one tube was coated with BSA to determine the specificity of the RNA-protein interaction. The last two tubes were coated with either the recombinant A18 hnRNP protein or nucleolin, another RNA-binding protein known for its specific binding to stem loop RNA structure (16Ghisolfi-Nieto L. Joseph G. Puvion-Dutilleul F. Amalric F. Bouvet P. J. Mol. Biol. 1996; 260: 34-53Crossref PubMed Scopus (164) Google Scholar). The data presented in Fig. 2 indicate that, under all the elution conditions used, the amount of RNA bound nonspecifically to BSA or the tube walls was less than 20% of the RNA bound to either A18 hnRNP or nucleolin. To make sure that we had eluted all the protein-bound RNA, we treated the tubes with proteinase K. As shown in Fig. 2 (lanes 17–20), proteinase K treatment released some RNA in all the tubes but in much smaller amounts than the salt treatment. These data indicate that under the conditions used most RNAs that are specifically bound to an RNA-binding protein can be eluted. The immunotube technique is thus a suitable and selective technique to identify the potential targets of a given RNA-binding protein. We then repeated this technique with a newly coated A18 hnRNP immunotube and 10 μg of mRNAs isolated from UV-irradiated RKO cells. Because the RKO cells contain several RBP that can be activated by genotoxic stress (7Carrier F. Gatignol A. Hollander M.C. Jeang K.T. Fornace Jr., A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1554-1558Crossref PubMed Scopus (25) Google Scholar), we reasoned that UV-irradiated RKO cells could be a good source of mRNA targets for A18 hnRNP. After incubation of the UV-treated mRNAs with the A18 hnRNP-coated tube, the specifically bound mRNAs were precipitated, reverse-transcribed, and amplified by PCR. The resulting amplicons were cloned and digested with the four-base cutter HaeIII (New England Biolabs, Beverly, MA) to evaluate redundancy. Based on this analysis we have determined that several genes were present more than once in the library. Sequencing was performed randomly on non-redundant clones by an automated sequencer. The partial sequences were compared with GenBankTM and EST data bases through the BLAST search engine. As shown in Table I, three main classes of transcripts representing 46 different clones have been identified. A large proportion (∼40%) of these genes are UV- or stress-responsive. This indicates that the technique is highly selective.Table ImRNAs selected by A18 hnRNPRibosomal proteinsStress-responsive proteinsOthersL3Replication protein A2 (43)18 S rRNAL5 (20)Thioredoxin (38)Novel sequence 1L10 (21)Ferritin L chain (41)Sequence of unknown functionL13A (24)Nucleophosmin (44)HistoneL15Glutathione transferase Ω (45)USF1 geneL17Translational elongation factor 1α (46)cctd1-aChaperonin containing T-complex.L19 (36)TIM1-bTranslocase of inner mitochondrial membrane. 22 homolog (47)HSPC023 mRNAL29Cyclophilin B (48)Novel sequence 2L30NADH-ubiquinone oxidoreductase (49)Hypothetical protein FLJ20420L32Human Wilm's tumor related (50)Tubulin-αS3aMyosin regulatory light chain (51)Cystein-glycine-rich protein CSRP2S5Laminin-binding protein (52)S6 (23)Human G10 homolog edg-2 (53)S8Translational elongation factor 1β2 (46)S11Protein kinase C inhibitor I (54)S12Putative C-myc-responsive (rcl) (55)S13 (22)ATPase (56)TAXREB1071-cDNA-binding protein. (57)References for stress-responsive proteins are given in parentheses.1-a Chaperonin containing T-complex.1-b Translocase of inner mitochondrial membrane.1-c DNA-binding protein. Open table in a new tab References for stress-responsive proteins are given in parentheses. Four of these genes, RPA2, thioredoxin, ferritin, and nucleophosmin, are either UV-inducible or UV-responsive (17Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (400) Google Scholar). We also verified the specificity of the mRNAs selected by attempting to amplify by reverse transcription-PCR other transcripts known to be UV-inducible. Transcripts for the growth arrest and DNA damage-inducible geneGADD45 and the cyclin-dependent kinase inhibitor p21 (1Fornace Jr., A.J. Jackman J. Hollander M.C. Hoffman-Liebermann B. Liebermann D.A. Ann. N. Y. Acad. Sci. 1992; 663: 139-153Crossref PubMed Scopus (174) Google Scholar) were not found in the pool of mRNAs selected by A18 hnRNP under these conditions. Another major class of transcripts bound by A18 hnRNP encodes ribosomal proteins. This large number of ribosomal proteins is in good agreement with the increasing body of evidence indicating that translation is an important component of the cellular stress response (18Sheikh M.S Fornace Jr., A.J. Oncogene. 1999; 18: 6121-6128Crossref PubMed Scopus (104) Google Scholar). Several type of stress, including heat shock stress and several chemicals, can induce the synthesis of stress proteins while inhibiting the rate of protein synthesis (Ref. 19Duncan R.F. Hershey J.W. Arch. Biochem. Biophys. 1987; 256: 651-661Crossref PubMed Scopus (33) Google Scholar and references within). Stress-induced changes in the stoichiometry of ribosomal proteins may trigger an adaptive response by allowing the translation of a specific set of proteins (20Bertram J. Palfner K. Hiddemann W. Kneba M. Eur. J. Cancer. 1998; 34: 731-736Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Three of the ribosomal proteins identified in our screen, L10, S13, and L19, have been associated with the oxidative stress response (21Mendez-Alvarez S. Rufenacht K. Eggen R.I. Biochem. Biophys. Res. Commun. 2000; 267: 953-959Crossref PubMed Scopus (25) Google Scholar, 22Dubnau E. Soares S. Huang T.J. Jacobs Jr., W.R. Gene. 1996; 170: 17-22Crossref PubMed Scopus (8) Google Scholar). L5 is involved in cellular resistance to general stress stimulus (20Bertram J. Palfner K. Hiddemann W. Kneba M. Eur. J. Cancer. 1998; 34: 731-736Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and S6 is induced by cold shock (23Graumann P. Schroder K. Schmid R. Marahiel M.A. J. Bacteriol. 1996; 178: 4611-4619Crossref PubMed Google Scholar). L13 has been suggested as a mediator of the stress induction of the sigma (B) factor in bacteria (24Scott J.M. Ju J. Mitchell T. Haldenwang W.G. J. Bacteriol. 2000; 182: 2771-2777Crossref PubMed Scopus (96) Google Scholar). These data support the idea that our technique is highly selective and that A18 hnRNP specifically targets transcripts involved in the stress response. We do not know whether the other ribosomal proteins identified in our screen play a direct or indirect role in the stress response. Nevertheless, A18 hnRNP may contribute to modulate an adaptive response to cellular stress by targeting specific sets of ribosomal protein transcripts. Other genes encoding a variety of transcripts were also identified. Among these, two encode new sequences, one of unknown function and one that encodes a hypothetical protein. Binding of A18 hnRNP to the 18 S rRNA is also of interest since this rRNA is part of the 40 S ribosomal small subunit. Binding to this rRNA may thus indicate a potential role for A18 hnRNP in translation. Based on sequence analysis we have determined that the 18 S rRNA sequence bound to A18 hnRNP corresponds to nucleotides 706–1385 of the 18 S rRNA (data not shown). This sequence is located in the central domain of the 18 S rRNA and is thought to be involved in eIF3 interaction, which prevents premature association of the large and small ribosomal subunits (25Melander Y. Holmberg L. Nygard O. J. Biol. Chem. 1997; 272: 3254-3258Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). A potential role in translation regulation is also supported by the binding of A18 hnRNP to the transcripts of two translational elongation factors (1α and 1β2). Our immunotube technique revealed that A18 hnRNP binds to a large number of stress-responsive transcripts (Table I). To determine the binding specificity of A18 hnRNP, we performed Northwestern analyses with RPA2 and thioredoxin, two stress-responsive transcripts identified in our screen (Table I). RPA2 and thioredoxin were selected for this assay based on their UV responsiveness and their well established role in the stress response. The RPA2 protein is important for DNA replication and nucleotide excision repair and is specifically phosphorylated after exposure to UV radiation (17Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (400) Google Scholar). RPA2 is also implicated in UV-induced replication arrest (17Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (400) Google Scholar). Thioredoxin is a UV-inducible protein involved in transcriptional processes such as induction of AP-1 activity and the inhibition of NF-κB activation (26Powis G. Mustacich D. Coon A. Free Radic. Biol. Med. 2000; 29: 312-322Crossref PubMed Scopus (374) Google Scholar). Thioredoxin has also been associated with tumor growth and is now becoming a new target for anti-cancer drugs (26Powis G. Mustacich D. Coon A. Free Radic. Biol. Med. 2000; 29: 312-322Crossref PubMed Scopus (374) Google Scholar). We first constructed four overlapping probes with the RPA2 transcript (Fig. 3 A) and hybridized them to increasing amounts of recombinant A18 hnRNP protein (Fig. 3 B). Our data indicate that A18 hnRNP does not bind to RPA2 ORF or the 5′-UTR. On the other hand, A18 hnRNP binds very strongly to RPA2 3′-UTR irrespective of the presence of the poly(A) tail. Binding is detectable with as little as 200 ng of recombinant protein (Fig. 3 B, lanes 2). We also repeated a similar experiment with the thioredoxin transcript (Fig. 4). Six different overlapping probes were generated (Fig. 4 A). Our data (Fig. 4 B) indicate that again A18 hnRNP does not bind to the ORF of the transcript and binds only weakly to the 5′-UTR. However, in this case, binding is reinforced by the presence of the transcript poly(A) tail. Interestingly, binding appears to be more sensitive in the presence of the ORF even though A18 hnRNP does not bind to it. The presence of the ORF may affect the overall structure of the RNA and increase binding. These data con
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