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Characterization of the 5′‐untranslated region of YB‐1 mRNA and autoregulation of translation by YB‐1 protein

Characterization of the 5′‐untranslated region of YB‐1 mRNA and autoregulation of translation by... Published online January 29, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 2 611±622 DOI: 10.1093/nar/gkh223 Characterization of the 5¢-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein 1 2 1 1 1 Takao Fukuda , Megumi Ashizuka , Takanori Nakamura , Kotaro Shibahara , 2 3 3 1 4 Katsumasa Maeda , Hiroto Izumi , Kimitoshi Kohno , Michihiko Kuwano and Takeshi Uchiumi * Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, 3-1-1 Maidashi, Fukuoka 812±8582, Japan, Section of Periodontology, Division of Oral Rehabilitation, Graduate School of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-0054, Japan, Department of Molecular Biology, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu 807-8555, Japan and Research Center for Innovative Cancer Therapy, Kurume University, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan Received August 16, 2003; Revised November 6, 2003; Accepted December 17, 2003 ABSTRACT prokaryotes and eukaryotes and are characterized by the evolutionary conservation of a cold shock domain (CSD). The eukaryotic Y-box binding protein YB-1 is Recently, it was reported that a major protein component of involved in various biological processes, including messenger ribonucleoprotein (mRNP) particles in somatic DNA repair, cell proliferation and the regulation of cells is a member of the Y-box binding transcription factor transcription and translation. YB-1 protein is abun- family. This protein acts either as a repressor or an activator of dant and expressed ubiquitously in human cells, protein synthesis (1±4). It has been hypothesized that YB-1 functioning in cell proliferation and transformation. might play a role in promoting cell proliferation through the Its concentration is thought to be highly regulated transcriptional regulation of various genes, including epider- at both the levels of transcription and translation. mal growth factor receptor, thymidine kinase, DNA topo- isomerase II and DNA polymerase (5,6). The multiple Therefore, we investigated whether or not the biological roles of YB-1 include the modi®cation of 5¢-UTR of YB-1 mRNA affects the translation of YB-1 chromatin, the translational masking of mRNA, participation protein, thus in¯uencing expression levels. in a redox signaling pathway, RNA chaperoning and regula- Luciferase mRNA ligated to the YB-1 mRNA 5¢-UTR tion of the stress response (7). was used as a reporter construct. Ligation of the It has also been demonstrated that eukaryotic Y-box full-length YB-1 5¢-UTR (331 bases) enhanced trans- proteins regulate gene expression at the level of translation lation as assessed by in vitro and in vivo translation by binding directly to RNA (8,9). The rabbit Y-box protein, assays. Deletion constructs of the YB-1 5¢-UTR also p50, is found in cytoplasmic mRNP particles in somatic cells resulted in a higher ef®ciency of translation, espe- and regulates translation by interacting with mRNA (2). The cially in the region mapped to +197 to +331 from the murine MSY1 protein and chicken Y-box protein both major transcription start site. RNA gel shift assays regulate transcription and translation (7,10±12). revealed that the af®nity of YB-1 for various 5¢-UTR Furthermore, the Y-box family proteins, Xenopus mRNP3/ probe sequences was higher for the full-length mRNP4 and mouse MSY2, have also been found to be 5¢-UTR than for deleted 5¢-UTR sequences. An mRNA-masking proteins in germinal cells (13±15). Chen et al. (16) have reported that YB-1 is involved in the mRNA in vitro translation assay was used to demonstrate stability of the cdk4 gene; this stability is achieved by the that recombinant YB-1 protein inhibited translation binding of YB-1 to a speci®c sequence of the mRNA. YB-1 of the full-length 5¢-UTR of YB-1 mRNA. Thus, our also stabilizes cap-dependent mRNA, since depletion of YB-1 ®ndings provide evidence for the autoregulation of results in accelerated mRNA decay (17). YB-1 mRNA translation via the 5¢-UTR. Previously, we identi®ed several proteins as partners of YB- 1, including YB-1 itself, iron regulatory protein 2 (IRP2) and the ribosomal proteins S3A, L18A, L5, L23A and S5. We also INTRODUCTION provided evidence that YB-1 is involved in the translational Y-box proteins function as transcriptional and translational regulation of an iron-related protein (18). Y-box binding regulators of gene expression. They are found among proteins thus appear to perform critical functions in both *To whom correspondence should be addressed. Tel: +81 92 642 6098; Fax: +81 92 642 6203; Email: [email protected] Nucleic Acids Research, Vol. 32 No. 2 ã Oxford University Press 2004; all rights reserved 612 Nucleic Acids Research, 2004, Vol. 32, No. 2 mRNA turnover and translational control. Considering the ampli®ed by PCR using the complementary primer pair. The important cellular functions of YB-1, it is possible that its YB-1 5¢-UTR-ligated luciferase cDNA fragment was cloned expression is highly regulated in eukaryotes. In fact, the YB-1 into the EcoRI-digested pT7Blue3 vector in order to generate gene behaves like a primary response gene. Stimulation of plasmid pT7-YB5¢-1. To functionally characterize the 5¢-UTR mammalian cell proliferation in culture or in vivo results in of the human YB-1 gene, a series of 5¢-deletion plasmids (pT7- increased YB-1 synthesis. The cellular level of YB-1 is YB5¢-2±pT7-YB5¢-6) were ampli®ed by PCR using the pT7- usually controlled by regulating the translation of its mRNA. It YB5¢-1 plasmid as a template. The forward primers were 5¢- is thought that an increase in the cellular YB-1 concentration AAGGTCCAATGAGAATGGAGGA-3¢ (pT7-YB5¢-2), 5¢- could alter the translation and stability of some mRNAs. AAGCTAGGGATTGGGGTCAG-3¢ (pT7-YB5¢-3), 5¢-CCT- Therefore, several pathways exist to control the function of AGGGCGGGTCGCTCGTA-3¢ (pT7-YB5¢-4), 5¢-CGATCG- this important cellular protein. GTAGCGGGAGCGGAG-3¢ (pT7-YB5¢-5) and 5¢-CCG- The 5¢- and 3¢-untranslated regions (UTRs) of eukaryotic CCGCCGCCGGCC-3¢ (pT7-YB5¢-6). Each of the PCR- mRNAs are known to play a crucial role in post-transcrip- ampli®ed fragments were cloned into the EcoRI-digested tional regulation that modulates nucleo-cytoplasmic mRNA pT7Blue3 vector to generate the pT7-YB5¢-2±pT7-YB5¢-6 transport, translation ef®ciency, subcellular localization and plasmids. To construct pCMV and pCMV-YB5¢-1±pCMV- stability (19). Several regulatory signals have already been YB5¢-6 plasmids suitable for expression in mammalian cells, identi®ed within the 5¢-or3¢-UTR sequences (20). These the pT7 or pT7-YB5¢-1±pT7-YB5¢-6 plasmids were digested signals tend to correspond to short oligonucleotide tracts, able with EcoRI. The fragments of luciferase cDNA were ligated to fold into speci®c secondary structures which provide into the EcoRI-digested pIRES-EYFP vector (Clontech, Palo binding sites for various regulatory proteins (21±23). Alto, CA), using various sizes of the YB-1 5¢-UTR region To examine how YB-1 mRNA translation is regulated in (pCMV-YB5¢-1±pCMV-YB5¢-6); a non-ligated fragment eukaryotic cells, we examined the possible role of its (pCMV) was used as a control. relatively long 5¢-UTR. Deletion of the YB-1 mRNA 5¢- Site-directed mutagenesis of the YB-1 5¢-UTR in pT7- UTR enhances translational activity in both in vitro and in vivo YB5¢-1 was performed using a PCR-based method. To obtain systems. The af®nities of YB-1 for 5¢-UTR probe sequences of pT7-YBM1±pT7-YBM3, the full length of the YB-1 5¢-UTR various lengths were evaluated by RNA gel shift assays; the sequence was ®rst ampli®ed by PCR. The forward primers af®nity of YB-1 was higher for the full-length 5¢-UTR than for were 5¢-GGTGGGCAGTACATCAGTACCACTGG-3¢ (pT7- truncated sequences. The addition of recombinant YB-1 YBM1), 5¢-GCGGGTCGCTAGAGAGGCTTATCCCGC-3¢ inhibited translation through the 5¢-UTR of its mRNA; this (pT7-YBM2), 5¢-CATTCTCGCTAGAACAGTCGGTAG- effect was particularly marked when the full-length 5¢-UTR CGGG-3¢ (pT7-YBM3) and the reverse primers were 5¢- was used. In this study, we have demonstrated for the ®rst time CCAGTGGTACTGATGTACTGCCCACC-3¢ (pT7-YBM1), that the 5¢-UTR region of human YB-1 mRNA plays an 5¢GCGGGATAAGCCTCTCTAGCGACCCGC-3¢ (pT7- important role in determining the conditions of YB-1 YBM2) and 5¢-CCCGCTACCGACTGTTCTAGCGAGA- biosynthesis at the translational level. ATG-3¢ (pT7-YBM3). A second PCR was then performed with Taq polymerase using the ®rst PCR products as templates. The PCR products were cloned into the EcoRI- digested pT7Blue3 vector in order to generate the pT7- MATERIALS AND METHODS YBM1±pT7-YBM3 plasmids. All constructs were con®rmed Construction of fusion protein expression plasmids by sequencing using a DNA sequencing system (model 377; Applied Biosystems, Foster City, CA). The plasmids containing full-length glutathione S-transferase (GST)±YB-1 cDNA fusions, GST±YB-1 deletion mutants and Cell lines Flag±YB-1 were described previously (24±26). A human epidermoid cancer cell line, KB3-1, was cultured in Reporter gene constructs MEM supplemented with 10% newborn calf serum. The A pT7 control plasmid, for in vitro transcription and human lung cancer cell line H1299 was cultured in RPMI translation experiments, was constructed by digesting lucifer- supplemented with 10% fetal bovine serum (FBS). HUVECs ase cDNA of a pGL3 basic vector (Promega, Madison, WI) were isolated from individual human umbilical cord veins by with EcoRI, blunting with Klenow enzyme, and ligation to collagenase digestion and were routinely cultured on type 1 pT7Blue3 (Novagen, Madison, WI). The pT7-YB5¢-1 plasmid collagen-coated plates in endothelial cell growth medium was constructed as follows. The entire length of the YB-1 5¢- (Clonetics, Boston, MA) supplemented with 2% FBS. Tissue UTR was ampli®ed by PCR from human YB-1 cDNA. The samples were obtained under an Institutional Review Board forward primer was 5¢-AGGCAGGAACGGTTGTAGGT-3¢ approved protocol, after the subjects had provided informed and the reverse primer was 5¢-gtttttggcgtcttccat- consent. The cells were maintained under standard cell culture GGTTGCGGTGATGG-3¢. The latter contains a luciferase conditions at 37°C and 5% CO in a humid environment. coding sequence at the 5¢-end (shown in lower case). A Recombinant proteins and antibodies luciferase cDNA fragment was also ampli®ed by PCR from a pGL3 basic vector, using the forward primer 5¢- Recombinant proteins were expressed in Escherichia coli CCATCACCGCAACCatggaagacgccaaaaac-3¢, complemen- DH5a. YB-1 and YB-1 deletion mutants were puri®ed as GST tary to the reverse primer of the YB-1 5¢-UTR and the reverse fusion proteins as described previously (25). Brie¯y, GST primer 5¢-ttacacggcgatctttcc-3¢. Each PCR-ampli®ed fragment fusion protein expressed in bacteria was induced by incubation was ligated with the complementary primer regions and with isopropyl-1-thio-b-D-galactopyranoside and cells were Nucleic Acids Research, 2004, Vol. 32, No. 2 613 lysed by sonication in 1 ml of binding buffer [1 mM ampli®cation products were analyzed by 2% agarose gel ditiothreitol, 0.5 mM phenylmethylsulfonyl ¯uoride (PMSF), electrophoresis. 200 mM NaCl, 10% v/v glycerol, 1% Triton-X, in phosphate- RNA band shift assays buffered saline (PBS) pH 7.3]. Cellular debris was removed by centrifugation and the supernatants were subjected to af®nity The RNA electrophoretic mobility shift assay (REMSA) was column chromatography using glutathione±Sepharose 4B carried out according to established techniques (29). Brie¯y, (Amersham Biosciences, Piscataway, NJ) according to the P-labeled YB-1 5¢-UTR probe was transcribed in vitro from manufacturer's recommendations. Antibody to YB-1 was the plasmid pT7blue3, which contains a sequence corres- generated as described previously (27). ponding to the 5¢-UTR of YB-1. An aliquot of 2 mg of the linearized plasmid was transcribed in vitro by T7 RNA Primer extension by reverse transcriptase polymerase in the presence of [a- P]UTP. The DNA template was removed by digestion with DNase I and the YB-1 5¢-UTR The primer extension experiments were carried out as probe was then extracted by column chromatography. To form described previously (28). Total RNA was prepared from RNA±protein complexes, 1±10 mg of cytoplasmic protein or each cell line using an RNeasy Miniprep Kit (Qiagen, the indicated amount of puri®ed GST±YB-1 was incubated Chatsworth, CA) and a QIAshredder microspin homogenizer with P-labeled YB-1 5¢-UTR probe at 25°C for 15 min. Next, according to the manufacturer's recommendations. The the samples underwent electrophoresis through a 4% poly(A) RNA was isolated from the total RNA using a non-denaturing polyacrylamide gel (polyacrylamide:bisacryl- Poly(A) Isolation Kit from Total RNA (Nippon Gene Co. amide, 80:1) in Tris±borate buffer. For the supershift experi- Ltd, Tokyo, Japan). The primer for the primer extension ments, 2 mg of the YB-1 antibody was incubated with analysis, 5¢-GCTCATGGTTGCGGTGATGG-3¢, was synthe- cytoplasmic protein or puri®ed GST±YB-1 at 25°C for 5 min sized to hybridize the sense strand between nucleotides ±14 before adding the P-labeled YB-1 5¢-UTR probe. The gels and +6 in the ®rst exon of the YB-1 gene. The synthetic primer were dried, visualized and then quanti®ed as described above was labeled at its 5¢-end with [g- P]ATP using T4 for the primer extension analysis. polynucleotide kinase and hybridized to 1 mg poly(A) RNA in 80% formamide, 0.4 M NaCl, 40 mM PIPES (pH 6.4) and In vitro transcription and translation 1 mM EDTA for 4 h at 50°C. After precipitation, the nucleic acid pellet was dissolved in reverse transcriptase buffer An aliquot of 2 mg of plasmid pT7Blue3, which encodes both (Invitrogen Corp., Carlsbad, CA). The primer was extended luciferase cDNA ligated to the YB-1 mRNA 5¢-UTR region with 200 U of SuperScript II RNase H reverse transcriptase (YB-1 5¢-UTR±luciferase) as well as luciferase cDNA not (Invitrogen) using 1 mM each of the four deoxynucleotides. ligated to the YB-1 mRNA 5¢-UTR region (luciferase) (see After 1 h at 37°C, the reaction was neutralized and the DNA Fig. 2A), was transcribed in vitro using an In vitro was collected. The reaction products were analyzed on a 7 M Transcription System (Promega). The DNA template was urea±8% polyacrylamide gel to determine the size of the removed by digestion with DNase I and the RNA was puri®ed extended product. The gel was exposed to an imaging plate by phenol/chloroform extraction. The integrity of the RNA and the blots were visualized and quanti®ed using a was then examined using an Agilent 2000 Bioanalizer phosphorimaging analyzer (model BAS 2000; Fuji Photo (Yokogawa, Osaka, Japan). Then, 50 ng of each RNA was Film Co., Tokyo, Japan) and the Image Gauge (version 3.4) translated using a rabbit reticulocyte lysate system (Promega). program. The luciferase assay was performed after incubation for 1.5 h at 30°C as described previously (18). To characterize the RNA immunoprecipitation assay translation inhibition of each of the reporter constructs, the indicated amounts of GST fusion proteins were added to Cells (100 mm dishes) were transfected with 5 mg of Flag±YB- the rabbit reticulocyte lysate system either initially or after 1 plasmid DNA using LipofectAMINE 2000 reagent 10 min of incubation using 50 ng of the RNA constructs. The (Invitrogen). After 48 h, the cell extract was preincubated in vitro translation was performed for 30 min at 30°C and the with protein A/G±agarose in TNE buffer [50 mM Tris±HCl translation activity of each experiment was measured. Data (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, are shown as the means 6 SD from three independent 1 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin] for 1 h experiments. at 4°C with rotation. After centrifugation, the supernatant was incubated with anti-Flag M2-agarose af®nity gel (Sigma Northern blot analysis Chemical Co., St Louis, MO) for 12 h at 4°C with rotation in TNE buffer. The beads were washed four times with TNE To detect in vitro transcribed RNA, a luciferase cDNA was buffer. After centrifugation, total RNA was extracted from the used as the probe. The luciferase cDNA was obtained by precipitate using a RNeasy Miniprep Kit (Qiagen). Total digesting the pGL3-Basic vector (Promega) with NarI and mRNAs were reverse transcribed and ampli®ed by PCR using XbaI. Reaction mixtures (20 ml, as described above) contain- the ThermoScript RT±PCR System (Invitrogen). The follow- ing 50 ng of in vitro transcribed RNA were preincubated or ing YB-1- and b-actin-speci®c primer pairs were used: the treated after 10 min incubation with 5 pmol of GST±YB-1. forward primers were 5¢-ACCACAGTATTCCAACCC- After incubation for 30 min in a rabbit reticulocyte lysate TCCTG-3¢ (YB-1) and 5¢-CTGGCACCACACCTTCTA- system, total RNA was extracted using an RNeasy Miniprep CAATG-3¢ (b-actin) and the reverse primers were 5¢-ATC- Kit (Qiagen). RNA samples (0.5 mg/lane) were separated on a TTCTTCATTGCCGTCCTCTC-3¢ (YB-1) and 5¢-ATAGC- 1% formaldehyde±agarose gel and transferred onto Biodyne B AACGTACATGGCTGGGG-3¢ (b-actin). The RT±PCR membranes (Pall, Port Washington, NY). The membrane was 614 Nucleic Acids Research, 2004, Vol. 32, No. 2 prehybridized and hybridized with [a- P]dCTP-labeled probe. Radioactivity was analyzed by autoradiograpy. Transfections and luciferase assays Cells underwent transient transfection using the LipofectAMINE method. Human lung cancer H1299 cells were plated at a density of 1 3 10 cells/35 mm well the day before transfection. At ~80±90% con¯uence, the cells were transfected with 1 mg of reporter plasmid DNA or control vector using LipofectAMINE 2000 reagent (Invitrogen). Three hours later, the cells were washed twice with PBS and placed in fresh medium. Twenty-four hours post-transfection, the luciferase activity was measured as described below. The luciferase activity of the transfected cells was measured using the Dual Luciferase Assay System (Promega). Brie¯y, cells were lysed with 250 mlof13 Passive Lysis Buffer (Promega). After a brief centrifugation, 10 ml of each supernatant was assayed for luciferase activity. Light emission was measured for 15 s with a luminometer. To standardize translation ef®ciency, the relative luciferase activity was expressed as the ratio of downstream cistron expression to upstream cistron expression (luciferase/EYFP). The ¯uorescence of enhanced yellow ¯uorescent protein (EYFP) was excited at 488 nm and measured for 1 s using a ¯uorometer. RESULTS Analysis of the multiple transcription initiation sites of the YB-1 gene To investigate the mechanisms of translational control regu- lating YB-1 protein levels, the possible involvement of YB-1 mRNA was investigated. The 5¢-UTR has a high G+C content (61%), suggesting that it could assume a high level of Figure 1. Analysis of the transcription start site of the YB-1 gene by primer extension assay. (A) Primer extension assay. Hybridization of the primer secondary structure in vivo. Computer modeling of potential extension was performed with 5¢-labeled oligonucleotide and 1 mg poly(A) secondary structures suggested that structures with free RNA of each cell line. The markers shown on the left are end-labeled HinfI energies (DG values) lower than ±190 kcal/mol could be fragments of f-X174 DNA. Black asterisks (*) indicate transcription formed (data not shown). We previously identi®ed several initiation start sites. The 5¢ furthest transcription initiation start site is indicated as +1 and the ®rst AUG codon is indicated at +331. The arrow transcription initiation sites for the YB-1 gene in KB3-1 and indicates the position of the primer that was used for extension. The primer T24 cells (28). To compare the major transcription initiation hybridizes the sense strand between nucleotides +317 and +337 of the YB-1 sites in other cell lines, primer extension analysis was gene. (B) Schematic distribution of the transcription initiation site in the performed (Fig. 1A). The lung cancer cell line H1299 and YB-1 5¢-UTR. endothelial cells (HUVECs), were compared with KB3-1 cells. The transcription initiation sites (*) were identical in all of the cells and eight transcripts (*1±*8) were observed in the designated +1 in the text and ®gures. Additional experiments region mapped to +1 to +197 (Fig. 1B). The ratio of the were performed to investigate the regulatory region of the 5¢- transcripts differed in each cell line. The proportion of UTR of YB-1 mRNA. A total of ®ve serial deletion mutants transcripts starting at initiation site *1 was signi®cantly higher (pT7-YB-5¢-2±pT7-YB5¢-6) were constructed, each corres- in HUVECs, compared to the other cancer cells. These results ponding to a particular transcription initiation site. We ®rst suggest that the multiple transcription start sites of YB-1 cloned transcripts *1, *2, *4, *7 and *8, which were each mRNA observed in cell lines may be involved in the expressed at up to 10% of the total. These transcripts regulation of YB-1 protein expression. corresponded to YB5¢-1±YB5¢-5. We made an additional probe, YB5¢-6, for detection of the shortest 5¢-UTR fragment The 5¢-UTR of YB-1 mRNA increases the expression of of YB-1. In vitro transcription reactions were performed on a luciferase reporter in vitro and in vivo each of these constructs using T7 RNA polymerase and the To study the possible involvement of the 5¢-UTR of YB-1 integrity of the transcripts was con®rmed by gel electrophor- mRNA in translation control, we generated two types of esis (data not shown). The differences in the transcript sizes reporter constructs. (i) Those containing luciferase cDNA were consistent with the length of the 5¢-UTR (Fig. 2A). Equal ligated to the full-length 5¢-UTR of YB-1 gene (pT7-YB5¢-1) amounts of the transcripts were translated in rabbit reticulo- and (ii) the non-ligated luciferase control construct (pT7) cyte lysate and the luciferase activity was measured. The (Fig. 2A). The predominant 5¢ transcription initiation site is presence of the full-length 5¢-UTR of YB-1 mRNA by itself Nucleic Acids Research, 2004, Vol. 32, No. 2 615 Figure 2. Effect of deletions of the 5¢-UTR of YB-1 on the expression of a luciferase reporter in vitro and in vivo. (A) Schematic representation of the reporter constructs containing luciferase cDNA ligated to the YB-1 5¢-UTR region (pT7-YB5¢-1±pT7-YB5¢-6) fragments of various sizes, as well as the non- ligated control construct (pT7). The YB-1 5¢-UTR region (pT7-YB5¢-1±pT7-YB5¢-6) fragments were enlarged to show the limits of regions of the 5¢-UTR of YB-1. The right-angled arrow denotes the start site and direction of transcription. Each of the reporter plasmids was transcribed in vitro in a reaction driven by T7 RNA polymerase and then 50 ng of the RNA constructs were translated using the rabbit reticulocyte lysate system. The luciferase assay was performed after incubation for 90 min at 30°C. (B) Schematic representation of the bicistronic reporter constructs containing luciferase cDNA ligated to the YB-1 5¢- UTR region (pCMV-YB5¢-1±pCMV-YB5¢-6) fragments of various sizes, as well as the non-ligated control construct (pCMV). Each of the reporter plasmids was transcribed under the control of the human cytomegalovirus early promoter (CMV). The right-angled arrow indicates the start site and direction of trans- lation. H1299 cells were transfected with these constructs and their luciferase activities were measured. To standardize the translation ef®ciency, relative luci- ferase activity was expressed as the ratio of downstream cistron expression to upstream cistron expression (luciferase/EYFP). Data are shown as the means 6 SD (error bars) of three independent experiments. increased the level of luciferase activity ~2-fold relative to that by the reporter constructs were equally expressed, as deter- of the control mRNA (pT7). Furthermore, each of the YB-1 5¢- mined by northern blot analysis (data not shown). These UTR deletion constructs showed higher translational activity results suggest that the YB-1 5¢-UTR enables more ef®cient than did the pT7 control. Of all the constructs, pT7-YB5¢-5 translation of mRNA in vitro and in vivo and the short-length construct (YB5¢-5) facilitates the most ef®cient translational showed the highest activity, which was ~4-fold greater than activity. that of the control construct (Fig. 2A). We next determined whether a similar increase in activity YB-1 binds its own mRNA in the cytoplasm could also be induced in cultured cells by the 5¢-UTR. We constructed eukaryotic expression vectors containing lucifer- YB-1, which posseses RNA binding activity (26), has been ase cDNA ligated to various regions of the YB-1 5¢-UTR reported to be involved in translational regulation and in the (pCMV-YB5¢-1±pCMV-YB5¢-6), as described for the in vitro regulation of mRNA stability (16). In order to identify whether experiment (Fig. 2A). The reporter constructs were transcribed or not YB-1 protein interacts with its own mRNA in the under control of the human cytomegalovirus early promoter. cytoplasm, we performed RT±PCR using mRNA isolated by After transfection into a H1299 lung carcinoma cell line, the co-immunoprecipitation with YB-1 immunoprecipitant levels of luciferase activity were compared (Fig. 2B). As (Fig. 3). Flag±YB-1 or an empty Flag expression vector observed with the in vitro translation assays (Fig. 2A), the YB- were transfected into an H1299 lung carcinoma cell line. After 15¢-UTR increased the level of luciferase expression. The 48 h, cells were lysed and YB-1 proteins were immunopre- full-length YB-1 5¢-UTR by itself increased luciferase activity cipitated with Flag antibody. The mRNA was puri®ed after ~2-fold compared to the control construct (pCMV). immunoprecipitation and ampli®ed by RT±PCR using YB-1- Furthermore, pCMV-YB5¢-5 also showed the highest activity, and b-actin-speci®c primers. YB-1 mRNA and b-actin mRNA which was 4±5-fold that of the control. The mRNAs encoded were both expressed in the H1299 cell lines (Fig. 3, lane 4). 616 Nucleic Acids Research, 2004, Vol. 32, No. 2 Figure 3. YB-1 interacts with its own mRNA in the cytoplasm. Flag±YB-1 or Flag expression vector was transfected into H1299 cells. After 48 h, cells were lysed and the YB-1 proteins were immunoprecipitated (IP) with Flag antibody. The mRNA were puri®ed after immunoprecipitation and ampli®ed by RT±PCR with YB-1- (upper panel) and b-actin-speci®c (lower panel) primers. The RT±PCR ampli®cation products were analyzed by 2% agarose gel electrophoresis. Flag±YB-1 immunoprecipitates contained YB-1 mRNA (Fig. 3, lane 3), while the control Flag immnoprecipitate did not (Fig. 3, lane 2). Furthermore, the YB-1 immunoprecipitate also contained b-actin mRNA (Fig. 3, lane 3). These data provide evidence that YB-1 interacts with its own mRNA and b-actin mRNA in the cytoplasm of cultured cells. YB-1 binds to the 5¢-UTR of its cognate mRNA through a C-terminal domain We next investigated the interaction of YB-1 protein with the 5¢-UTR of its own mRNA in vitro. An RNA gel shift assay (REMSA) was performed by using KB3-1 and H1299 cell lysates and an in vitro synthesized mRNA probe correspond- ing to the full-length 5¢-UTR of YB-1 mRNA (Fig. 4A). YB-1 5¢-UTR formed an RNA±protein complex with lysates made using KB3-1 and H1299 cell lysates (Fig. 4A, lanes 2±4 and 8±10). The presence of endogenous YB-1 protein in the major complex was con®rmed by the ability of a YB-1-speci®c antibody to supershift most of the complex (Fig. 4A, lanes 6 and 12). To determine whether YB-1 protein is able to directly interact with the 5¢-UTR of YB-1 mRNA, we performed a REMSA using puri®ed recombinant YB-1 (Fig. 4B). Figure 4. YB-1 binds to the 5¢-UTR region of YB-1mRNA. (A) Endogenous Recombinant YB-1 also clearly bound to the 5¢-UTR of YB-1 protein binds to the 5¢-UTR region of YB-1 mRNA in the cytoplasm. YB-1 mRNA (Fig. 4B, lanes 5±7), while control GST protein The indicated amounts of KB3-1 and H1299 cell lysate were incubated with did not (Fig. 4B, lanes 2±4). The interaction with YB-1 protein P-labeled YB-1 5¢-UTR RNA at 25°C for 15 min. An aliquot of 2 mg YB-1- was also con®rmed by the use of YB-1-speci®c antibody speci®c antibody (Ab) was added to lanes 5, 6, 11 and 12. An arrow indicates (Fig. 4B, lanes 10 and 11). the YB-1/YB-1 5¢-UTR RNA complex and a double-headed arrow indicates supershifted complexes. (B) Puri®ed YB-1 binds to YB-1 5¢-UTR RNA. The We previously observed that rabbit p50, a homolog of indicated amount of GST or GST±YB-1 fusion was incubated with P-labeled human YB-1 protein, was present in rabbit reticulocyte lysate. YB-1 5¢-UTR RNA at 25°C for 15 min. An aliquot of 2 mg YB-1-speci®c anti- To assess the nature of the rabbit p50 interaction with the 5¢- body (Ab) was added to lanes 8±11. An arrow indicates the YB-1/YB-1 5¢- UTR of YB-1 mRNA, we performed a REMSA using rabbit UTR RNA complex and a double-headed arrow indicates supershifted com- plexes. (C) Rabbit YB-1 (p50) binds to the 5¢-UTR region of YB-1 mRNA in reticulocyte lysate (Fig. 4C). When added to rabbit reticulo- the in vitro translation system. The indicated amounts of rabbit reticulocyte cyte lysate, the YB-1 5¢-UTR formed an RNA±protein lysate (RRL) were incubated with P-labeled YB-1 5¢-UTR RNA at 25°C for complex (Fig. 4C, lanes 2 and 3). The presence of rabbit 15 min. An aliquot of 2 mg YB-1-speci®c antibody (Ab) was added to lanes 4 p50 in the major complex was con®rmed by the ability of a and 5. An arrow indicates the rabbit YB-1/YB-1 5¢-UTR RNA complex and a YB-1-speci®c antibody to supershift most of the complex double-headed arrow indicates supershifted complexes. YB-1/YB-1 5¢-UTR RNA probe complexes were separated as described above. (Fig. 4C, lane 5). Nucleic Acids Research, 2004, Vol. 32, No. 2 617 Figure 5. Identi®cation of the YB-1 5¢-UTR binding domain in YB-1. (A) Schematic illustration of the GST±YB-1 deletion mutants used in this study. CSD indicates the cold shock domain. (B) REMSA assay. The indicated amount of each GST±YB-1 deletion mutant and GST was incubated with P-labeled YB-1 5¢-UTR RNA at 25°C for 15 min. An aliquot of 2 mg YB-1-speci®c antibody (Ab) was added to lanes 11±15. An arrow indicates the YB-1/YB-1 5¢-UTR RNA complex and a double-headed arrow indicates supershifted complexes. YB-1 mutant/YB-1 5¢-UTR RNA probe complexes were separated using 4% native polyacrylamide gels. YB-1 protein consists of three major domains, each of Figure 6. Identi®cation of the YB-1 binding region in YB-1 5¢-UTR which has the potential to bind nucleic acids (30): the alanine/ mRNA. (A) Schematic illustration of YB-1 5¢-UTR deletion constructs used proline-rich N-terminal domain, the highly conserved nucleic as the probe in a REMSA. To produce RNA of de®ned length, restriction enzyme (PvuI or PvuII) was used to linearize the DNA templates. acid binding domain and the C-tail domain. To identify which (B) REMSA. The indicated amount of GST±YB-1 fusion was incubated domains of YB-1 protein are responsible for this interaction, with each P-labeled YB-1 5¢-UTR deletion mRNA at 25°C for 15 min. An we performed a REMSA using a series of GST fusion proteins arrow indicates the YB-1/YB-1 5¢-UTR RNA complex. YB-1/YB-1 5¢-UTR containing either full-length YB-1 (FL) or the mutant RNA probe complexes were separated using 4% native polyacrylamide gels. derivatives GST±YB-1 N-ter, CSD and C-tail (Fig. 5A). A (C) Kinetic analysis of GST±YB-1 binding to YB5¢-1, YB/PvuI and YB5¢-5 probes. The GST±YB-1 binding activity to YB-1 5¢-UTR fragments strong interaction was observed using the full-length YB-1 identical to that shown in (B) were quanti®ed by monitoring amounts of the (Fig. 5B, lanes 3 and 4) and the C-tail of YB-1 (Fig. 5B, lanes binding complexes. 9 and 10). The central CSD, containing the RNP1 motif, was not able to bind to the 5¢-UTR of YB-1 mRNA (Fig. 5B, lanes corresponding to YB5¢-1 (295 bases), YB/PvuI (200 bases) 7 and 8). The binding activity of both constructs resulted in the and YB5¢-5 (98 bases), as de®ned in Figure 1B (Fig. 6A). appearance of a supershifted band in the presence of a YB-1- All three constructs formed RNA±protein complexes in a speci®c antibody that recognizes the C-terminal tail of YB-1 dose-dependent manner. However, the af®nity of YB-1 for the (Fig. 5B, lanes 12 and 15). These results suggest that the C-tail three probes differed. When GST±YB-1 was added to the full- domain of YB-1 protein interacts with the 5¢-UTR of its own length YB-1 5¢-UTR (YB5¢-1) and YB/PvuI, a retarded mRNA, independent of other domains. complex was clearly visible (Fig. 6B). When 0.6 pmol of GST±YB-1 was added, up to 60% of the YB5¢-1 and YB/PvuI Identi®cation of the YB-1 binding region in the 5¢-UTR probes were bound to GST±YB-1 (Fig. 6C). However, little of YB-1 mRNA retarded band was observed using the deleted YB5¢-5 probe To identify which region of the YB-1 mRNA 5¢-UTR interacts (Fig. 6B) when the same amount of YB-1 was used. When with YB-1 protein, we performed a REMSA using probes 0.6 pmol of YB-1 was added to YB5¢-5, <20% YB5¢-5 was 618 Nucleic Acids Research, 2004, Vol. 32, No. 2 bound. These results indicate that YB-1 protein binds to the full-length and the ®rst half of the YB-1 5¢-UTR more ef®ciently than a construct containing the second half of the YB-1 5¢-UTR, YB5¢-5. Recombinant YB-1 protein decreases translation through its own 5¢-UTR mRNA element in vitro As shown in the REMSA, we observed higher af®nity binding to the full-length 5¢-UTR of YB-1 mRNA with recombinant YB-1. It has been shown that inhibitory concentrations of YB- 1 suppressed the interaction with initiation complexes such as eIF4E, eIF4A and eIF4B, and these concentrations inhibited translation at the stage of initiation (17,31). YB-1 has also + + been reported to affect the translation of both cap poly(A) ± ± and cap poly(A) mRNAs (1,2). Skabkina et al. also demonstrated that poly(A) binding protein positively affects YB-1 mRNA translation through speci®c interaction with YB- 1 mRNA (32). Thus, we investigated whether or not recombinant YB-1 inhibits translation in a cell-free translation system by using the full-length and deleted YB-1 5¢-UTR constructs. In this system the transcript of YB-1 5¢-UTR were neither capped nor polyadenylated. We therefore excluded the possibility that binding between YB-1 protein and translation initiation and elongation factors such as cap binding protein or poly(A) binding protein was involved. To test the functional activity of YB-1 protein, recombinant YB-1 protein was added to the in vitro translation system. In vitro transcripts from pT7, pT7-YB5¢-1 and pT7-YB5¢-5 were translated in a rabbit reticulocyte lysate system in the absence or presence of recombinant YB-1 protein and the luciferase activity was measured. As shown in Figure 7A, the addition of YB-1 at the start of translation inhibited the translation of all three constructs in a dose-dependent manner to a maximum of ~60%. Next, we measured the kinetics of luciferase translation in a rabbit retilculocyte lysate. After the ®rst 10 min, almost 10% of translation had occurred and translation ef®ciency was saturated after 60 min (data not shown). To investigate the inhibitory effects of YB-1 protein on translation, we added various amounts of YB-1 protein 10 min after translation was Figure 7. Characterization of translation inhibition by YB-1 protein through started (Fig. 7B). The addition of YB-1 10 min after its own 5¢-UTR mRNA element in vitro. The indicated amounts of GST± translation inhibited the luciferase activity in a dose-depend- YB-1 fusion were added to a rabbit reticulocyte lysate system either initially ent manner when the construct containing the full-length 5¢- (A) or after 10 min incubation (B) using 50 ng of the RNA constructs de- scribed in Figure 2A. The in vitro translation was performed for 30 min at UTR (pT7-YB5¢-1) was used; this reduction amounted to 30°C and the translational products were directly used for the luciferase ~50% with 5 pmol of YB-1, implying that only 10% of the assay. Data are shown as means 6 SD (error bars) of three independent inhibition results from translation that could have occurred in experiments. (C) The effect of YB-1 on mRNA stability was examined. An the ®rst 10 min of the 30 min reaction. On the other hand, little aliquot of 50 ng of in vitro transcribed RNA constructs was preincubated YB-1-dependent repression of translation was seen using pT7- (pre) or treated after 10 min incubation (after) with 5 pmol of GST±YB-1. After incubation for 30 min in a rabbit reticulocyte lysate system, the RNA YB5¢-5 and pT7-luciferase. These results suggested that was detected by northern blot hybridization. Lanes 1, 4 and 7 (Input) show pretreated YB-1 inhibited translation of all constructs and the in vitro transcribed RNA constructs isolated from the exact at 0 min the addition of YB-1 after translation also speci®cally incubation. inhibited translation of the construct with the full-length 5¢- UTR. YB-1 protein was also involved in stabilization of mRNA in Recombinant YB-1 protein did not decrease translation rabbit reticulocyte system. We con®rmed the integrity of the through a mutant 5¢-UTR element in vitro transcribed mRNA by northern blot analysis before Next, we focused on the possible secondary structure of the and after treatment with YB-1 (Fig. 7C). No change in full-length 5¢-UTR of YB-1 mRNA. In general, the stem±loop transcribed RNA stability was observed with up to 30 min incubation in rabbit reticulocyte system (Fig. 7C, lanes 1±3, 4± structure found in 5¢-UTRs of mRNAs affect their transla- 6 and 7±9). tional ef®ciency as, for example, in the ferritin 5¢-UTR (18). In Nucleic Acids Research, 2004, Vol. 32, No. 2 619 the YB-1 5¢-UTR, we observed three regions which are predicted to form possible stem±loop structures. To investi- gate the translational regulatory region of the 5¢-UTR of YB-1 mRNA, we constructed three mutants which disrupt the stem± loop structure. These contained internal mutations (mut1± mut3) which we have designated using the nucleotide at the 5¢ end corresponding to the transcription initiation site (*) (Fig. 8A). To characterize the translational inhibition observed with the addition of YB-1, we performed cell-free trans- lational assays using mutants of the above constructs of the YB-1 5¢-UTR fused to luciferase mRNA (Fig. 8B and C). The addition of YB-1 at the start of translation resulted in up to a 60% inhibition of luciferase activity in a dose-dependent manner, in the case of both the mutant and full-length 5¢-UTR (Fig. 8B). However, no repression of the luciferase activity of these mutant constructs (pT7-YBM1±pT7-YBM3) was ob- served when YB-1 was added 10 min after translation was initiated (Fig. 8C). These results suggest that YB-1 protein regulates the translation of all constructs and also that it speci®cally inhibits translation when the 5¢-UTR of YB-1 mRNA is present. Our ®ndings are consistent with a model in which YB-1 protein speci®cally binds to the full-length 5¢- UTR of YB-1 mRNA and inhibits its own translation in vitro. DISCUSSION In this report, our data demonstrate that YB-1 protein not only binds to an RNA containing the human YB-1 5¢-UTR in vitro and in vivo, but that it also exhibits functional activity by speci®cally inhibiting the translation of a YB-1 5¢-UTR± luciferase reporter mRNA in rabbit reticulocyte lysate assays. The multifunctional protein YB-1 was ®rst identi®ed as a transcription factor which binds to the Y-box of the MHC class II promoter sequence (33). Recent studies have demon- strated that YB-1 regulates gene expression not only at the level of transcription, but also at the level of mRNA translation. Considering these characteristics, it is possible that YB-1 might regulate its own expression at the transla- tional level. The present paper provides evidence that the translational control of YB-1 expression is mediated via the 5¢-UTR of YB-1 mRNA. Global control of translational ef®ciency can be achieved by regulating the phosphorylation state of an array of initiation factors (such as eIF2 and eIF4E). However, control of translational initiation on an individual mRNA is deter- mined primarily by its nucleotide sequence and the secondary structure of the regulatory protein. The 5¢-UTR plays a particularly important role in the regulation of translation initiation via an interaction with RNA binding proteins or by secondary structure formation, which hinders the activity of the translational machinery. Analysis of the 5¢-UTR sequence Figure 8. Effect of mutation of the YB-1 5¢-UTR mRNA element on translation. (A) Schematic representation of the YB-1 5¢-UTR±luciferase of YB-1 (331 bases in length) revealed several important fusion reporter constructs used in this study. pT7-YBM1~3 containing an features that are known to in¯uence translation. internal mutation (mut1-3), designated by the nucleotide at the 5¢ end that Many in vitro and in vivo studies have shown that mRNAs corresponded to the transcription initiation sites (*) (see Fig. 1A). (B and C) with a high likelihood of forming a stable secondary structure The characterization of translation inhibition by each reporter construct was in the 5¢-UTR tend to be inef®ciently translated (34). compared to that of the other constructs. The indicated amounts of GST±YB-1 fusion were added to a rabbit reticulocyte lysate system either However, in both in vitro and in vivo experiments we found initially (B) or after 10 min incubation (C) using 50 ng of the RNA that the 5¢-UTR sequence of YB-1 mRNA, with its high G+C constructs described in (A). The in vitro translation was performed for content, acts as a potent translational enhancer. The full-length 30 min at 30°C and the translation activity of each experiment was meas- 5¢-UTR of YB-1 mRNA increased the level of translation ured. Data are shown as the means 6 SD (error bars) of three independent experiments. activity and cloning a 134 base region of the YB-1 5¢-UTR 620 Nucleic Acids Research, 2004, Vol. 32, No. 2 upstream of the coding initiation sequence was also associated YB-1 has been reported to be involved in the regulation of with a signi®cant increase in translation activity (Fig. 2A and stress-inducible target genes (40,41), suggesting that YB-1 B). This region might therefore be crucial for the translation itself is a stress-activated protein. YB-1 mRNA accumulates activity of the 5¢-UTR of YB-1 mRNA. These data suggest in cells when they are treated with UV irradiation or that elements within the 5¢-UTR of YB-1 mRNA can act as anticancer agents. We previously reported that c-myc and enhancers of mRNA translation. p73 activate YB-1 transcription and may regulate important According to the scanning mechanism postulated for biological processes via their effects on YB-1 gene expression translation initiation, the small (40S) ribosomal subunit enters (42). Additional experiments are needed to con®rm the role of at the 5¢ end of the mRNA and migrates linearly, stopping YB-1 in regulating translation in vivo under various stress when the ®rst AUG codon is reached (35). Nekrasov et al. (31) conditions. It would be of interest to determine how the 5¢- demonstrated that YB-1 protein inhibited the initiation step of UTR of YB-1 mRNA affects the translation of YB-1 in translation, but did not inhibit the elongation step. Rabbit YB- response to extracellular stimuli or environmental stress. 1 displays both RNA melting and annealing activities in a The translational control of gene expression has been dose-dependent manner; a relatively low amount of YB-1 identi®ed as an important regulatory mechanism for many promotes the formation of RNA duplexes, whereas an excess gene products involved in the regulation of proliferation [e.g. of YB-1 causes unwinding of double-stranded forms (36). It is c-mos (43), FGF-2 (44,45), PDGF-B (46), p27 (47) and cdk4 also possible that YB-1 additionally facilitates movement of (48)]. The tumor suppressor p53 has been implicated in the the small ribosomal subunit to the initiation codon complexed translational regulation of both p53 and cdk4 mRNAs (49). Met with initiator tRNA . This may occur up to the initiation Similarly, several distinct components of the translation codon by YB-1 melting the secondary structure in the mRNA apparatus have been shown to be deregulated or overexpressed in human tumors. So, it is of note that the level of the YB-1 5¢-UTR (31). mRNA from the 5¢ furthest transcription initiation start site In this study, we observed that YB-1 inhibited the was signi®cantly higher in HUVEC cells, compared to the translation of all of the transcripts of YB-1 5¢-UTR that other cancer cell lines examined (Fig. 1). The deregulation of lacked sequence speci®city, suggesting that YB-1 reduced the the translational control of YB-1 might therefore also play a translation ef®ciency of 5¢-UTR elements, either at the role in tumor progression. initiation step or by binding directly to the whole RNA. In conclusion, YB-1 protein binds to an RNA containing the These results provided evidence that YB-1 might block the 5¢-UTR of human YB-1 mRNA in vitro and in vivo and ®rst step in mRNA recruitment into translation initiation. exhibits functional activity by inhibiting the translation of a The addition of YB-1 10 min after initiation inhibited YB-1 5¢-UTR±luciferase reporter mRNA through the aid of its translational activity only when the full-length 5¢-UTR of YB- own 5¢-UTR mRNA. We propose a model in which YB-1 1 mRNA was present (Figs 7B and 8C). No repression of the protein autoregulates its own translation by binding to the 5¢- luciferase activity of the deleted or mutant constructs (pT7- YBM1±pT7-YBM3) (with disrupted stem±loop structures) UTR of its own mRNA. Our results support a trans-acting was observed. We also observed that YB-1 binds to the ®rst repressor hypothesis, in which a repressor protein speci®cally half-region of YB-1 5¢ UTR RNA more ef®ciently than does binds to its own 5¢-UTR element, resulting in translational the latter half construct comprising the second half (Fig. 7). repression and the maintenance of constant protein levels. The af®nity of YB-1 for the mutated probes was the same as for the wild-type YB5¢-1 probes (data not shown). These results suggest that ribosome scanning or ongoing translation ACKNOWLEDGEMENTS might be required for repression by YB-1 when the full-length We would like to thank Prof. R.G. Deeley for helpful YB-1 5¢-UTR was used. And when mutated and deleted probe discussions and for critically reading this manuscript. This were used, YB-1 did not affect ribosomal scanning due to work was supported by the Second-Term Comprehensive Ten- alterations in the secondary structure of the YB-1 5¢ UTR. The Year Strategy for Cancer Control from the Ministry of Health precise secondary structures of the YB-1 5¢-UTR remains to and Welfare of Japan and by the Cancer Research Fund from be determined. the Ministry of Education, Culture, Sports, Science and By combining the results presented here with previous data, Technology. we are able to describe the effect of YB-1 on translation. First, YB-1 blocks the initial step in mRNA recruitment to translation initiation, YB-1 then turns off the interaction of REFERENCES the mRNA with eIF4F (31). Second, YB-1 promotes move- ment of the small ribosomal subunit within the complex by 1. Pisarev,A.V., Skabkin,M.A., Thomas,A.A., Merrick,W.C., melting the mRNA 5¢-UTR secondary structure, suggesting Ovchinnikov,L.P. and Shatsky,I.N. 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Characterization of the 5′‐untranslated region of YB‐1 mRNA and autoregulation of translation by YB‐1 protein

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Oxford University Press
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10.1093/nar/gkh223
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Abstract

Published online January 29, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 2 611±622 DOI: 10.1093/nar/gkh223 Characterization of the 5¢-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein 1 2 1 1 1 Takao Fukuda , Megumi Ashizuka , Takanori Nakamura , Kotaro Shibahara , 2 3 3 1 4 Katsumasa Maeda , Hiroto Izumi , Kimitoshi Kohno , Michihiko Kuwano and Takeshi Uchiumi * Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, 3-1-1 Maidashi, Fukuoka 812±8582, Japan, Section of Periodontology, Division of Oral Rehabilitation, Graduate School of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-0054, Japan, Department of Molecular Biology, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu 807-8555, Japan and Research Center for Innovative Cancer Therapy, Kurume University, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan Received August 16, 2003; Revised November 6, 2003; Accepted December 17, 2003 ABSTRACT prokaryotes and eukaryotes and are characterized by the evolutionary conservation of a cold shock domain (CSD). The eukaryotic Y-box binding protein YB-1 is Recently, it was reported that a major protein component of involved in various biological processes, including messenger ribonucleoprotein (mRNP) particles in somatic DNA repair, cell proliferation and the regulation of cells is a member of the Y-box binding transcription factor transcription and translation. YB-1 protein is abun- family. This protein acts either as a repressor or an activator of dant and expressed ubiquitously in human cells, protein synthesis (1±4). It has been hypothesized that YB-1 functioning in cell proliferation and transformation. might play a role in promoting cell proliferation through the Its concentration is thought to be highly regulated transcriptional regulation of various genes, including epider- at both the levels of transcription and translation. mal growth factor receptor, thymidine kinase, DNA topo- isomerase II and DNA polymerase (5,6). The multiple Therefore, we investigated whether or not the biological roles of YB-1 include the modi®cation of 5¢-UTR of YB-1 mRNA affects the translation of YB-1 chromatin, the translational masking of mRNA, participation protein, thus in¯uencing expression levels. in a redox signaling pathway, RNA chaperoning and regula- Luciferase mRNA ligated to the YB-1 mRNA 5¢-UTR tion of the stress response (7). was used as a reporter construct. Ligation of the It has also been demonstrated that eukaryotic Y-box full-length YB-1 5¢-UTR (331 bases) enhanced trans- proteins regulate gene expression at the level of translation lation as assessed by in vitro and in vivo translation by binding directly to RNA (8,9). The rabbit Y-box protein, assays. Deletion constructs of the YB-1 5¢-UTR also p50, is found in cytoplasmic mRNP particles in somatic cells resulted in a higher ef®ciency of translation, espe- and regulates translation by interacting with mRNA (2). The cially in the region mapped to +197 to +331 from the murine MSY1 protein and chicken Y-box protein both major transcription start site. RNA gel shift assays regulate transcription and translation (7,10±12). revealed that the af®nity of YB-1 for various 5¢-UTR Furthermore, the Y-box family proteins, Xenopus mRNP3/ probe sequences was higher for the full-length mRNP4 and mouse MSY2, have also been found to be 5¢-UTR than for deleted 5¢-UTR sequences. An mRNA-masking proteins in germinal cells (13±15). Chen et al. (16) have reported that YB-1 is involved in the mRNA in vitro translation assay was used to demonstrate stability of the cdk4 gene; this stability is achieved by the that recombinant YB-1 protein inhibited translation binding of YB-1 to a speci®c sequence of the mRNA. YB-1 of the full-length 5¢-UTR of YB-1 mRNA. Thus, our also stabilizes cap-dependent mRNA, since depletion of YB-1 ®ndings provide evidence for the autoregulation of results in accelerated mRNA decay (17). YB-1 mRNA translation via the 5¢-UTR. Previously, we identi®ed several proteins as partners of YB- 1, including YB-1 itself, iron regulatory protein 2 (IRP2) and the ribosomal proteins S3A, L18A, L5, L23A and S5. We also INTRODUCTION provided evidence that YB-1 is involved in the translational Y-box proteins function as transcriptional and translational regulation of an iron-related protein (18). Y-box binding regulators of gene expression. They are found among proteins thus appear to perform critical functions in both *To whom correspondence should be addressed. Tel: +81 92 642 6098; Fax: +81 92 642 6203; Email: [email protected] Nucleic Acids Research, Vol. 32 No. 2 ã Oxford University Press 2004; all rights reserved 612 Nucleic Acids Research, 2004, Vol. 32, No. 2 mRNA turnover and translational control. Considering the ampli®ed by PCR using the complementary primer pair. The important cellular functions of YB-1, it is possible that its YB-1 5¢-UTR-ligated luciferase cDNA fragment was cloned expression is highly regulated in eukaryotes. In fact, the YB-1 into the EcoRI-digested pT7Blue3 vector in order to generate gene behaves like a primary response gene. Stimulation of plasmid pT7-YB5¢-1. To functionally characterize the 5¢-UTR mammalian cell proliferation in culture or in vivo results in of the human YB-1 gene, a series of 5¢-deletion plasmids (pT7- increased YB-1 synthesis. The cellular level of YB-1 is YB5¢-2±pT7-YB5¢-6) were ampli®ed by PCR using the pT7- usually controlled by regulating the translation of its mRNA. It YB5¢-1 plasmid as a template. The forward primers were 5¢- is thought that an increase in the cellular YB-1 concentration AAGGTCCAATGAGAATGGAGGA-3¢ (pT7-YB5¢-2), 5¢- could alter the translation and stability of some mRNAs. AAGCTAGGGATTGGGGTCAG-3¢ (pT7-YB5¢-3), 5¢-CCT- Therefore, several pathways exist to control the function of AGGGCGGGTCGCTCGTA-3¢ (pT7-YB5¢-4), 5¢-CGATCG- this important cellular protein. GTAGCGGGAGCGGAG-3¢ (pT7-YB5¢-5) and 5¢-CCG- The 5¢- and 3¢-untranslated regions (UTRs) of eukaryotic CCGCCGCCGGCC-3¢ (pT7-YB5¢-6). Each of the PCR- mRNAs are known to play a crucial role in post-transcrip- ampli®ed fragments were cloned into the EcoRI-digested tional regulation that modulates nucleo-cytoplasmic mRNA pT7Blue3 vector to generate the pT7-YB5¢-2±pT7-YB5¢-6 transport, translation ef®ciency, subcellular localization and plasmids. To construct pCMV and pCMV-YB5¢-1±pCMV- stability (19). Several regulatory signals have already been YB5¢-6 plasmids suitable for expression in mammalian cells, identi®ed within the 5¢-or3¢-UTR sequences (20). These the pT7 or pT7-YB5¢-1±pT7-YB5¢-6 plasmids were digested signals tend to correspond to short oligonucleotide tracts, able with EcoRI. The fragments of luciferase cDNA were ligated to fold into speci®c secondary structures which provide into the EcoRI-digested pIRES-EYFP vector (Clontech, Palo binding sites for various regulatory proteins (21±23). Alto, CA), using various sizes of the YB-1 5¢-UTR region To examine how YB-1 mRNA translation is regulated in (pCMV-YB5¢-1±pCMV-YB5¢-6); a non-ligated fragment eukaryotic cells, we examined the possible role of its (pCMV) was used as a control. relatively long 5¢-UTR. Deletion of the YB-1 mRNA 5¢- Site-directed mutagenesis of the YB-1 5¢-UTR in pT7- UTR enhances translational activity in both in vitro and in vivo YB5¢-1 was performed using a PCR-based method. To obtain systems. The af®nities of YB-1 for 5¢-UTR probe sequences of pT7-YBM1±pT7-YBM3, the full length of the YB-1 5¢-UTR various lengths were evaluated by RNA gel shift assays; the sequence was ®rst ampli®ed by PCR. The forward primers af®nity of YB-1 was higher for the full-length 5¢-UTR than for were 5¢-GGTGGGCAGTACATCAGTACCACTGG-3¢ (pT7- truncated sequences. The addition of recombinant YB-1 YBM1), 5¢-GCGGGTCGCTAGAGAGGCTTATCCCGC-3¢ inhibited translation through the 5¢-UTR of its mRNA; this (pT7-YBM2), 5¢-CATTCTCGCTAGAACAGTCGGTAG- effect was particularly marked when the full-length 5¢-UTR CGGG-3¢ (pT7-YBM3) and the reverse primers were 5¢- was used. In this study, we have demonstrated for the ®rst time CCAGTGGTACTGATGTACTGCCCACC-3¢ (pT7-YBM1), that the 5¢-UTR region of human YB-1 mRNA plays an 5¢GCGGGATAAGCCTCTCTAGCGACCCGC-3¢ (pT7- important role in determining the conditions of YB-1 YBM2) and 5¢-CCCGCTACCGACTGTTCTAGCGAGA- biosynthesis at the translational level. ATG-3¢ (pT7-YBM3). A second PCR was then performed with Taq polymerase using the ®rst PCR products as templates. The PCR products were cloned into the EcoRI- digested pT7Blue3 vector in order to generate the pT7- MATERIALS AND METHODS YBM1±pT7-YBM3 plasmids. All constructs were con®rmed Construction of fusion protein expression plasmids by sequencing using a DNA sequencing system (model 377; Applied Biosystems, Foster City, CA). The plasmids containing full-length glutathione S-transferase (GST)±YB-1 cDNA fusions, GST±YB-1 deletion mutants and Cell lines Flag±YB-1 were described previously (24±26). A human epidermoid cancer cell line, KB3-1, was cultured in Reporter gene constructs MEM supplemented with 10% newborn calf serum. The A pT7 control plasmid, for in vitro transcription and human lung cancer cell line H1299 was cultured in RPMI translation experiments, was constructed by digesting lucifer- supplemented with 10% fetal bovine serum (FBS). HUVECs ase cDNA of a pGL3 basic vector (Promega, Madison, WI) were isolated from individual human umbilical cord veins by with EcoRI, blunting with Klenow enzyme, and ligation to collagenase digestion and were routinely cultured on type 1 pT7Blue3 (Novagen, Madison, WI). The pT7-YB5¢-1 plasmid collagen-coated plates in endothelial cell growth medium was constructed as follows. The entire length of the YB-1 5¢- (Clonetics, Boston, MA) supplemented with 2% FBS. Tissue UTR was ampli®ed by PCR from human YB-1 cDNA. The samples were obtained under an Institutional Review Board forward primer was 5¢-AGGCAGGAACGGTTGTAGGT-3¢ approved protocol, after the subjects had provided informed and the reverse primer was 5¢-gtttttggcgtcttccat- consent. The cells were maintained under standard cell culture GGTTGCGGTGATGG-3¢. The latter contains a luciferase conditions at 37°C and 5% CO in a humid environment. coding sequence at the 5¢-end (shown in lower case). A Recombinant proteins and antibodies luciferase cDNA fragment was also ampli®ed by PCR from a pGL3 basic vector, using the forward primer 5¢- Recombinant proteins were expressed in Escherichia coli CCATCACCGCAACCatggaagacgccaaaaac-3¢, complemen- DH5a. YB-1 and YB-1 deletion mutants were puri®ed as GST tary to the reverse primer of the YB-1 5¢-UTR and the reverse fusion proteins as described previously (25). Brie¯y, GST primer 5¢-ttacacggcgatctttcc-3¢. Each PCR-ampli®ed fragment fusion protein expressed in bacteria was induced by incubation was ligated with the complementary primer regions and with isopropyl-1-thio-b-D-galactopyranoside and cells were Nucleic Acids Research, 2004, Vol. 32, No. 2 613 lysed by sonication in 1 ml of binding buffer [1 mM ampli®cation products were analyzed by 2% agarose gel ditiothreitol, 0.5 mM phenylmethylsulfonyl ¯uoride (PMSF), electrophoresis. 200 mM NaCl, 10% v/v glycerol, 1% Triton-X, in phosphate- RNA band shift assays buffered saline (PBS) pH 7.3]. Cellular debris was removed by centrifugation and the supernatants were subjected to af®nity The RNA electrophoretic mobility shift assay (REMSA) was column chromatography using glutathione±Sepharose 4B carried out according to established techniques (29). Brie¯y, (Amersham Biosciences, Piscataway, NJ) according to the P-labeled YB-1 5¢-UTR probe was transcribed in vitro from manufacturer's recommendations. Antibody to YB-1 was the plasmid pT7blue3, which contains a sequence corres- generated as described previously (27). ponding to the 5¢-UTR of YB-1. An aliquot of 2 mg of the linearized plasmid was transcribed in vitro by T7 RNA Primer extension by reverse transcriptase polymerase in the presence of [a- P]UTP. The DNA template was removed by digestion with DNase I and the YB-1 5¢-UTR The primer extension experiments were carried out as probe was then extracted by column chromatography. To form described previously (28). Total RNA was prepared from RNA±protein complexes, 1±10 mg of cytoplasmic protein or each cell line using an RNeasy Miniprep Kit (Qiagen, the indicated amount of puri®ed GST±YB-1 was incubated Chatsworth, CA) and a QIAshredder microspin homogenizer with P-labeled YB-1 5¢-UTR probe at 25°C for 15 min. Next, according to the manufacturer's recommendations. The the samples underwent electrophoresis through a 4% poly(A) RNA was isolated from the total RNA using a non-denaturing polyacrylamide gel (polyacrylamide:bisacryl- Poly(A) Isolation Kit from Total RNA (Nippon Gene Co. amide, 80:1) in Tris±borate buffer. For the supershift experi- Ltd, Tokyo, Japan). The primer for the primer extension ments, 2 mg of the YB-1 antibody was incubated with analysis, 5¢-GCTCATGGTTGCGGTGATGG-3¢, was synthe- cytoplasmic protein or puri®ed GST±YB-1 at 25°C for 5 min sized to hybridize the sense strand between nucleotides ±14 before adding the P-labeled YB-1 5¢-UTR probe. The gels and +6 in the ®rst exon of the YB-1 gene. The synthetic primer were dried, visualized and then quanti®ed as described above was labeled at its 5¢-end with [g- P]ATP using T4 for the primer extension analysis. polynucleotide kinase and hybridized to 1 mg poly(A) RNA in 80% formamide, 0.4 M NaCl, 40 mM PIPES (pH 6.4) and In vitro transcription and translation 1 mM EDTA for 4 h at 50°C. After precipitation, the nucleic acid pellet was dissolved in reverse transcriptase buffer An aliquot of 2 mg of plasmid pT7Blue3, which encodes both (Invitrogen Corp., Carlsbad, CA). The primer was extended luciferase cDNA ligated to the YB-1 mRNA 5¢-UTR region with 200 U of SuperScript II RNase H reverse transcriptase (YB-1 5¢-UTR±luciferase) as well as luciferase cDNA not (Invitrogen) using 1 mM each of the four deoxynucleotides. ligated to the YB-1 mRNA 5¢-UTR region (luciferase) (see After 1 h at 37°C, the reaction was neutralized and the DNA Fig. 2A), was transcribed in vitro using an In vitro was collected. The reaction products were analyzed on a 7 M Transcription System (Promega). The DNA template was urea±8% polyacrylamide gel to determine the size of the removed by digestion with DNase I and the RNA was puri®ed extended product. The gel was exposed to an imaging plate by phenol/chloroform extraction. The integrity of the RNA and the blots were visualized and quanti®ed using a was then examined using an Agilent 2000 Bioanalizer phosphorimaging analyzer (model BAS 2000; Fuji Photo (Yokogawa, Osaka, Japan). Then, 50 ng of each RNA was Film Co., Tokyo, Japan) and the Image Gauge (version 3.4) translated using a rabbit reticulocyte lysate system (Promega). program. The luciferase assay was performed after incubation for 1.5 h at 30°C as described previously (18). To characterize the RNA immunoprecipitation assay translation inhibition of each of the reporter constructs, the indicated amounts of GST fusion proteins were added to Cells (100 mm dishes) were transfected with 5 mg of Flag±YB- the rabbit reticulocyte lysate system either initially or after 1 plasmid DNA using LipofectAMINE 2000 reagent 10 min of incubation using 50 ng of the RNA constructs. The (Invitrogen). After 48 h, the cell extract was preincubated in vitro translation was performed for 30 min at 30°C and the with protein A/G±agarose in TNE buffer [50 mM Tris±HCl translation activity of each experiment was measured. Data (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, are shown as the means 6 SD from three independent 1 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin] for 1 h experiments. at 4°C with rotation. After centrifugation, the supernatant was incubated with anti-Flag M2-agarose af®nity gel (Sigma Northern blot analysis Chemical Co., St Louis, MO) for 12 h at 4°C with rotation in TNE buffer. The beads were washed four times with TNE To detect in vitro transcribed RNA, a luciferase cDNA was buffer. After centrifugation, total RNA was extracted from the used as the probe. The luciferase cDNA was obtained by precipitate using a RNeasy Miniprep Kit (Qiagen). Total digesting the pGL3-Basic vector (Promega) with NarI and mRNAs were reverse transcribed and ampli®ed by PCR using XbaI. Reaction mixtures (20 ml, as described above) contain- the ThermoScript RT±PCR System (Invitrogen). The follow- ing 50 ng of in vitro transcribed RNA were preincubated or ing YB-1- and b-actin-speci®c primer pairs were used: the treated after 10 min incubation with 5 pmol of GST±YB-1. forward primers were 5¢-ACCACAGTATTCCAACCC- After incubation for 30 min in a rabbit reticulocyte lysate TCCTG-3¢ (YB-1) and 5¢-CTGGCACCACACCTTCTA- system, total RNA was extracted using an RNeasy Miniprep CAATG-3¢ (b-actin) and the reverse primers were 5¢-ATC- Kit (Qiagen). RNA samples (0.5 mg/lane) were separated on a TTCTTCATTGCCGTCCTCTC-3¢ (YB-1) and 5¢-ATAGC- 1% formaldehyde±agarose gel and transferred onto Biodyne B AACGTACATGGCTGGGG-3¢ (b-actin). The RT±PCR membranes (Pall, Port Washington, NY). The membrane was 614 Nucleic Acids Research, 2004, Vol. 32, No. 2 prehybridized and hybridized with [a- P]dCTP-labeled probe. Radioactivity was analyzed by autoradiograpy. Transfections and luciferase assays Cells underwent transient transfection using the LipofectAMINE method. Human lung cancer H1299 cells were plated at a density of 1 3 10 cells/35 mm well the day before transfection. At ~80±90% con¯uence, the cells were transfected with 1 mg of reporter plasmid DNA or control vector using LipofectAMINE 2000 reagent (Invitrogen). Three hours later, the cells were washed twice with PBS and placed in fresh medium. Twenty-four hours post-transfection, the luciferase activity was measured as described below. The luciferase activity of the transfected cells was measured using the Dual Luciferase Assay System (Promega). Brie¯y, cells were lysed with 250 mlof13 Passive Lysis Buffer (Promega). After a brief centrifugation, 10 ml of each supernatant was assayed for luciferase activity. Light emission was measured for 15 s with a luminometer. To standardize translation ef®ciency, the relative luciferase activity was expressed as the ratio of downstream cistron expression to upstream cistron expression (luciferase/EYFP). The ¯uorescence of enhanced yellow ¯uorescent protein (EYFP) was excited at 488 nm and measured for 1 s using a ¯uorometer. RESULTS Analysis of the multiple transcription initiation sites of the YB-1 gene To investigate the mechanisms of translational control regu- lating YB-1 protein levels, the possible involvement of YB-1 mRNA was investigated. The 5¢-UTR has a high G+C content (61%), suggesting that it could assume a high level of Figure 1. Analysis of the transcription start site of the YB-1 gene by primer extension assay. (A) Primer extension assay. Hybridization of the primer secondary structure in vivo. Computer modeling of potential extension was performed with 5¢-labeled oligonucleotide and 1 mg poly(A) secondary structures suggested that structures with free RNA of each cell line. The markers shown on the left are end-labeled HinfI energies (DG values) lower than ±190 kcal/mol could be fragments of f-X174 DNA. Black asterisks (*) indicate transcription formed (data not shown). We previously identi®ed several initiation start sites. The 5¢ furthest transcription initiation start site is indicated as +1 and the ®rst AUG codon is indicated at +331. The arrow transcription initiation sites for the YB-1 gene in KB3-1 and indicates the position of the primer that was used for extension. The primer T24 cells (28). To compare the major transcription initiation hybridizes the sense strand between nucleotides +317 and +337 of the YB-1 sites in other cell lines, primer extension analysis was gene. (B) Schematic distribution of the transcription initiation site in the performed (Fig. 1A). The lung cancer cell line H1299 and YB-1 5¢-UTR. endothelial cells (HUVECs), were compared with KB3-1 cells. The transcription initiation sites (*) were identical in all of the cells and eight transcripts (*1±*8) were observed in the designated +1 in the text and ®gures. Additional experiments region mapped to +1 to +197 (Fig. 1B). The ratio of the were performed to investigate the regulatory region of the 5¢- transcripts differed in each cell line. The proportion of UTR of YB-1 mRNA. A total of ®ve serial deletion mutants transcripts starting at initiation site *1 was signi®cantly higher (pT7-YB-5¢-2±pT7-YB5¢-6) were constructed, each corres- in HUVECs, compared to the other cancer cells. These results ponding to a particular transcription initiation site. We ®rst suggest that the multiple transcription start sites of YB-1 cloned transcripts *1, *2, *4, *7 and *8, which were each mRNA observed in cell lines may be involved in the expressed at up to 10% of the total. These transcripts regulation of YB-1 protein expression. corresponded to YB5¢-1±YB5¢-5. We made an additional probe, YB5¢-6, for detection of the shortest 5¢-UTR fragment The 5¢-UTR of YB-1 mRNA increases the expression of of YB-1. In vitro transcription reactions were performed on a luciferase reporter in vitro and in vivo each of these constructs using T7 RNA polymerase and the To study the possible involvement of the 5¢-UTR of YB-1 integrity of the transcripts was con®rmed by gel electrophor- mRNA in translation control, we generated two types of esis (data not shown). The differences in the transcript sizes reporter constructs. (i) Those containing luciferase cDNA were consistent with the length of the 5¢-UTR (Fig. 2A). Equal ligated to the full-length 5¢-UTR of YB-1 gene (pT7-YB5¢-1) amounts of the transcripts were translated in rabbit reticulo- and (ii) the non-ligated luciferase control construct (pT7) cyte lysate and the luciferase activity was measured. The (Fig. 2A). The predominant 5¢ transcription initiation site is presence of the full-length 5¢-UTR of YB-1 mRNA by itself Nucleic Acids Research, 2004, Vol. 32, No. 2 615 Figure 2. Effect of deletions of the 5¢-UTR of YB-1 on the expression of a luciferase reporter in vitro and in vivo. (A) Schematic representation of the reporter constructs containing luciferase cDNA ligated to the YB-1 5¢-UTR region (pT7-YB5¢-1±pT7-YB5¢-6) fragments of various sizes, as well as the non- ligated control construct (pT7). The YB-1 5¢-UTR region (pT7-YB5¢-1±pT7-YB5¢-6) fragments were enlarged to show the limits of regions of the 5¢-UTR of YB-1. The right-angled arrow denotes the start site and direction of transcription. Each of the reporter plasmids was transcribed in vitro in a reaction driven by T7 RNA polymerase and then 50 ng of the RNA constructs were translated using the rabbit reticulocyte lysate system. The luciferase assay was performed after incubation for 90 min at 30°C. (B) Schematic representation of the bicistronic reporter constructs containing luciferase cDNA ligated to the YB-1 5¢- UTR region (pCMV-YB5¢-1±pCMV-YB5¢-6) fragments of various sizes, as well as the non-ligated control construct (pCMV). Each of the reporter plasmids was transcribed under the control of the human cytomegalovirus early promoter (CMV). The right-angled arrow indicates the start site and direction of trans- lation. H1299 cells were transfected with these constructs and their luciferase activities were measured. To standardize the translation ef®ciency, relative luci- ferase activity was expressed as the ratio of downstream cistron expression to upstream cistron expression (luciferase/EYFP). Data are shown as the means 6 SD (error bars) of three independent experiments. increased the level of luciferase activity ~2-fold relative to that by the reporter constructs were equally expressed, as deter- of the control mRNA (pT7). Furthermore, each of the YB-1 5¢- mined by northern blot analysis (data not shown). These UTR deletion constructs showed higher translational activity results suggest that the YB-1 5¢-UTR enables more ef®cient than did the pT7 control. Of all the constructs, pT7-YB5¢-5 translation of mRNA in vitro and in vivo and the short-length construct (YB5¢-5) facilitates the most ef®cient translational showed the highest activity, which was ~4-fold greater than activity. that of the control construct (Fig. 2A). We next determined whether a similar increase in activity YB-1 binds its own mRNA in the cytoplasm could also be induced in cultured cells by the 5¢-UTR. We constructed eukaryotic expression vectors containing lucifer- YB-1, which posseses RNA binding activity (26), has been ase cDNA ligated to various regions of the YB-1 5¢-UTR reported to be involved in translational regulation and in the (pCMV-YB5¢-1±pCMV-YB5¢-6), as described for the in vitro regulation of mRNA stability (16). In order to identify whether experiment (Fig. 2A). The reporter constructs were transcribed or not YB-1 protein interacts with its own mRNA in the under control of the human cytomegalovirus early promoter. cytoplasm, we performed RT±PCR using mRNA isolated by After transfection into a H1299 lung carcinoma cell line, the co-immunoprecipitation with YB-1 immunoprecipitant levels of luciferase activity were compared (Fig. 2B). As (Fig. 3). Flag±YB-1 or an empty Flag expression vector observed with the in vitro translation assays (Fig. 2A), the YB- were transfected into an H1299 lung carcinoma cell line. After 15¢-UTR increased the level of luciferase expression. The 48 h, cells were lysed and YB-1 proteins were immunopre- full-length YB-1 5¢-UTR by itself increased luciferase activity cipitated with Flag antibody. The mRNA was puri®ed after ~2-fold compared to the control construct (pCMV). immunoprecipitation and ampli®ed by RT±PCR using YB-1- Furthermore, pCMV-YB5¢-5 also showed the highest activity, and b-actin-speci®c primers. YB-1 mRNA and b-actin mRNA which was 4±5-fold that of the control. The mRNAs encoded were both expressed in the H1299 cell lines (Fig. 3, lane 4). 616 Nucleic Acids Research, 2004, Vol. 32, No. 2 Figure 3. YB-1 interacts with its own mRNA in the cytoplasm. Flag±YB-1 or Flag expression vector was transfected into H1299 cells. After 48 h, cells were lysed and the YB-1 proteins were immunoprecipitated (IP) with Flag antibody. The mRNA were puri®ed after immunoprecipitation and ampli®ed by RT±PCR with YB-1- (upper panel) and b-actin-speci®c (lower panel) primers. The RT±PCR ampli®cation products were analyzed by 2% agarose gel electrophoresis. Flag±YB-1 immunoprecipitates contained YB-1 mRNA (Fig. 3, lane 3), while the control Flag immnoprecipitate did not (Fig. 3, lane 2). Furthermore, the YB-1 immunoprecipitate also contained b-actin mRNA (Fig. 3, lane 3). These data provide evidence that YB-1 interacts with its own mRNA and b-actin mRNA in the cytoplasm of cultured cells. YB-1 binds to the 5¢-UTR of its cognate mRNA through a C-terminal domain We next investigated the interaction of YB-1 protein with the 5¢-UTR of its own mRNA in vitro. An RNA gel shift assay (REMSA) was performed by using KB3-1 and H1299 cell lysates and an in vitro synthesized mRNA probe correspond- ing to the full-length 5¢-UTR of YB-1 mRNA (Fig. 4A). YB-1 5¢-UTR formed an RNA±protein complex with lysates made using KB3-1 and H1299 cell lysates (Fig. 4A, lanes 2±4 and 8±10). The presence of endogenous YB-1 protein in the major complex was con®rmed by the ability of a YB-1-speci®c antibody to supershift most of the complex (Fig. 4A, lanes 6 and 12). To determine whether YB-1 protein is able to directly interact with the 5¢-UTR of YB-1 mRNA, we performed a REMSA using puri®ed recombinant YB-1 (Fig. 4B). Figure 4. YB-1 binds to the 5¢-UTR region of YB-1mRNA. (A) Endogenous Recombinant YB-1 also clearly bound to the 5¢-UTR of YB-1 protein binds to the 5¢-UTR region of YB-1 mRNA in the cytoplasm. YB-1 mRNA (Fig. 4B, lanes 5±7), while control GST protein The indicated amounts of KB3-1 and H1299 cell lysate were incubated with did not (Fig. 4B, lanes 2±4). The interaction with YB-1 protein P-labeled YB-1 5¢-UTR RNA at 25°C for 15 min. An aliquot of 2 mg YB-1- was also con®rmed by the use of YB-1-speci®c antibody speci®c antibody (Ab) was added to lanes 5, 6, 11 and 12. An arrow indicates (Fig. 4B, lanes 10 and 11). the YB-1/YB-1 5¢-UTR RNA complex and a double-headed arrow indicates supershifted complexes. (B) Puri®ed YB-1 binds to YB-1 5¢-UTR RNA. The We previously observed that rabbit p50, a homolog of indicated amount of GST or GST±YB-1 fusion was incubated with P-labeled human YB-1 protein, was present in rabbit reticulocyte lysate. YB-1 5¢-UTR RNA at 25°C for 15 min. An aliquot of 2 mg YB-1-speci®c anti- To assess the nature of the rabbit p50 interaction with the 5¢- body (Ab) was added to lanes 8±11. An arrow indicates the YB-1/YB-1 5¢- UTR of YB-1 mRNA, we performed a REMSA using rabbit UTR RNA complex and a double-headed arrow indicates supershifted com- plexes. (C) Rabbit YB-1 (p50) binds to the 5¢-UTR region of YB-1 mRNA in reticulocyte lysate (Fig. 4C). When added to rabbit reticulo- the in vitro translation system. The indicated amounts of rabbit reticulocyte cyte lysate, the YB-1 5¢-UTR formed an RNA±protein lysate (RRL) were incubated with P-labeled YB-1 5¢-UTR RNA at 25°C for complex (Fig. 4C, lanes 2 and 3). The presence of rabbit 15 min. An aliquot of 2 mg YB-1-speci®c antibody (Ab) was added to lanes 4 p50 in the major complex was con®rmed by the ability of a and 5. An arrow indicates the rabbit YB-1/YB-1 5¢-UTR RNA complex and a YB-1-speci®c antibody to supershift most of the complex double-headed arrow indicates supershifted complexes. YB-1/YB-1 5¢-UTR RNA probe complexes were separated as described above. (Fig. 4C, lane 5). Nucleic Acids Research, 2004, Vol. 32, No. 2 617 Figure 5. Identi®cation of the YB-1 5¢-UTR binding domain in YB-1. (A) Schematic illustration of the GST±YB-1 deletion mutants used in this study. CSD indicates the cold shock domain. (B) REMSA assay. The indicated amount of each GST±YB-1 deletion mutant and GST was incubated with P-labeled YB-1 5¢-UTR RNA at 25°C for 15 min. An aliquot of 2 mg YB-1-speci®c antibody (Ab) was added to lanes 11±15. An arrow indicates the YB-1/YB-1 5¢-UTR RNA complex and a double-headed arrow indicates supershifted complexes. YB-1 mutant/YB-1 5¢-UTR RNA probe complexes were separated using 4% native polyacrylamide gels. YB-1 protein consists of three major domains, each of Figure 6. Identi®cation of the YB-1 binding region in YB-1 5¢-UTR which has the potential to bind nucleic acids (30): the alanine/ mRNA. (A) Schematic illustration of YB-1 5¢-UTR deletion constructs used proline-rich N-terminal domain, the highly conserved nucleic as the probe in a REMSA. To produce RNA of de®ned length, restriction enzyme (PvuI or PvuII) was used to linearize the DNA templates. acid binding domain and the C-tail domain. To identify which (B) REMSA. The indicated amount of GST±YB-1 fusion was incubated domains of YB-1 protein are responsible for this interaction, with each P-labeled YB-1 5¢-UTR deletion mRNA at 25°C for 15 min. An we performed a REMSA using a series of GST fusion proteins arrow indicates the YB-1/YB-1 5¢-UTR RNA complex. YB-1/YB-1 5¢-UTR containing either full-length YB-1 (FL) or the mutant RNA probe complexes were separated using 4% native polyacrylamide gels. derivatives GST±YB-1 N-ter, CSD and C-tail (Fig. 5A). A (C) Kinetic analysis of GST±YB-1 binding to YB5¢-1, YB/PvuI and YB5¢-5 probes. The GST±YB-1 binding activity to YB-1 5¢-UTR fragments strong interaction was observed using the full-length YB-1 identical to that shown in (B) were quanti®ed by monitoring amounts of the (Fig. 5B, lanes 3 and 4) and the C-tail of YB-1 (Fig. 5B, lanes binding complexes. 9 and 10). The central CSD, containing the RNP1 motif, was not able to bind to the 5¢-UTR of YB-1 mRNA (Fig. 5B, lanes corresponding to YB5¢-1 (295 bases), YB/PvuI (200 bases) 7 and 8). The binding activity of both constructs resulted in the and YB5¢-5 (98 bases), as de®ned in Figure 1B (Fig. 6A). appearance of a supershifted band in the presence of a YB-1- All three constructs formed RNA±protein complexes in a speci®c antibody that recognizes the C-terminal tail of YB-1 dose-dependent manner. However, the af®nity of YB-1 for the (Fig. 5B, lanes 12 and 15). These results suggest that the C-tail three probes differed. When GST±YB-1 was added to the full- domain of YB-1 protein interacts with the 5¢-UTR of its own length YB-1 5¢-UTR (YB5¢-1) and YB/PvuI, a retarded mRNA, independent of other domains. complex was clearly visible (Fig. 6B). When 0.6 pmol of GST±YB-1 was added, up to 60% of the YB5¢-1 and YB/PvuI Identi®cation of the YB-1 binding region in the 5¢-UTR probes were bound to GST±YB-1 (Fig. 6C). However, little of YB-1 mRNA retarded band was observed using the deleted YB5¢-5 probe To identify which region of the YB-1 mRNA 5¢-UTR interacts (Fig. 6B) when the same amount of YB-1 was used. When with YB-1 protein, we performed a REMSA using probes 0.6 pmol of YB-1 was added to YB5¢-5, <20% YB5¢-5 was 618 Nucleic Acids Research, 2004, Vol. 32, No. 2 bound. These results indicate that YB-1 protein binds to the full-length and the ®rst half of the YB-1 5¢-UTR more ef®ciently than a construct containing the second half of the YB-1 5¢-UTR, YB5¢-5. Recombinant YB-1 protein decreases translation through its own 5¢-UTR mRNA element in vitro As shown in the REMSA, we observed higher af®nity binding to the full-length 5¢-UTR of YB-1 mRNA with recombinant YB-1. It has been shown that inhibitory concentrations of YB- 1 suppressed the interaction with initiation complexes such as eIF4E, eIF4A and eIF4B, and these concentrations inhibited translation at the stage of initiation (17,31). YB-1 has also + + been reported to affect the translation of both cap poly(A) ± ± and cap poly(A) mRNAs (1,2). Skabkina et al. also demonstrated that poly(A) binding protein positively affects YB-1 mRNA translation through speci®c interaction with YB- 1 mRNA (32). Thus, we investigated whether or not recombinant YB-1 inhibits translation in a cell-free translation system by using the full-length and deleted YB-1 5¢-UTR constructs. In this system the transcript of YB-1 5¢-UTR were neither capped nor polyadenylated. We therefore excluded the possibility that binding between YB-1 protein and translation initiation and elongation factors such as cap binding protein or poly(A) binding protein was involved. To test the functional activity of YB-1 protein, recombinant YB-1 protein was added to the in vitro translation system. In vitro transcripts from pT7, pT7-YB5¢-1 and pT7-YB5¢-5 were translated in a rabbit reticulocyte lysate system in the absence or presence of recombinant YB-1 protein and the luciferase activity was measured. As shown in Figure 7A, the addition of YB-1 at the start of translation inhibited the translation of all three constructs in a dose-dependent manner to a maximum of ~60%. Next, we measured the kinetics of luciferase translation in a rabbit retilculocyte lysate. After the ®rst 10 min, almost 10% of translation had occurred and translation ef®ciency was saturated after 60 min (data not shown). To investigate the inhibitory effects of YB-1 protein on translation, we added various amounts of YB-1 protein 10 min after translation was Figure 7. Characterization of translation inhibition by YB-1 protein through started (Fig. 7B). The addition of YB-1 10 min after its own 5¢-UTR mRNA element in vitro. The indicated amounts of GST± translation inhibited the luciferase activity in a dose-depend- YB-1 fusion were added to a rabbit reticulocyte lysate system either initially ent manner when the construct containing the full-length 5¢- (A) or after 10 min incubation (B) using 50 ng of the RNA constructs de- scribed in Figure 2A. The in vitro translation was performed for 30 min at UTR (pT7-YB5¢-1) was used; this reduction amounted to 30°C and the translational products were directly used for the luciferase ~50% with 5 pmol of YB-1, implying that only 10% of the assay. Data are shown as means 6 SD (error bars) of three independent inhibition results from translation that could have occurred in experiments. (C) The effect of YB-1 on mRNA stability was examined. An the ®rst 10 min of the 30 min reaction. On the other hand, little aliquot of 50 ng of in vitro transcribed RNA constructs was preincubated YB-1-dependent repression of translation was seen using pT7- (pre) or treated after 10 min incubation (after) with 5 pmol of GST±YB-1. After incubation for 30 min in a rabbit reticulocyte lysate system, the RNA YB5¢-5 and pT7-luciferase. These results suggested that was detected by northern blot hybridization. Lanes 1, 4 and 7 (Input) show pretreated YB-1 inhibited translation of all constructs and the in vitro transcribed RNA constructs isolated from the exact at 0 min the addition of YB-1 after translation also speci®cally incubation. inhibited translation of the construct with the full-length 5¢- UTR. YB-1 protein was also involved in stabilization of mRNA in Recombinant YB-1 protein did not decrease translation rabbit reticulocyte system. We con®rmed the integrity of the through a mutant 5¢-UTR element in vitro transcribed mRNA by northern blot analysis before Next, we focused on the possible secondary structure of the and after treatment with YB-1 (Fig. 7C). No change in full-length 5¢-UTR of YB-1 mRNA. In general, the stem±loop transcribed RNA stability was observed with up to 30 min incubation in rabbit reticulocyte system (Fig. 7C, lanes 1±3, 4± structure found in 5¢-UTRs of mRNAs affect their transla- 6 and 7±9). tional ef®ciency as, for example, in the ferritin 5¢-UTR (18). In Nucleic Acids Research, 2004, Vol. 32, No. 2 619 the YB-1 5¢-UTR, we observed three regions which are predicted to form possible stem±loop structures. To investi- gate the translational regulatory region of the 5¢-UTR of YB-1 mRNA, we constructed three mutants which disrupt the stem± loop structure. These contained internal mutations (mut1± mut3) which we have designated using the nucleotide at the 5¢ end corresponding to the transcription initiation site (*) (Fig. 8A). To characterize the translational inhibition observed with the addition of YB-1, we performed cell-free trans- lational assays using mutants of the above constructs of the YB-1 5¢-UTR fused to luciferase mRNA (Fig. 8B and C). The addition of YB-1 at the start of translation resulted in up to a 60% inhibition of luciferase activity in a dose-dependent manner, in the case of both the mutant and full-length 5¢-UTR (Fig. 8B). However, no repression of the luciferase activity of these mutant constructs (pT7-YBM1±pT7-YBM3) was ob- served when YB-1 was added 10 min after translation was initiated (Fig. 8C). These results suggest that YB-1 protein regulates the translation of all constructs and also that it speci®cally inhibits translation when the 5¢-UTR of YB-1 mRNA is present. Our ®ndings are consistent with a model in which YB-1 protein speci®cally binds to the full-length 5¢- UTR of YB-1 mRNA and inhibits its own translation in vitro. DISCUSSION In this report, our data demonstrate that YB-1 protein not only binds to an RNA containing the human YB-1 5¢-UTR in vitro and in vivo, but that it also exhibits functional activity by speci®cally inhibiting the translation of a YB-1 5¢-UTR± luciferase reporter mRNA in rabbit reticulocyte lysate assays. The multifunctional protein YB-1 was ®rst identi®ed as a transcription factor which binds to the Y-box of the MHC class II promoter sequence (33). Recent studies have demon- strated that YB-1 regulates gene expression not only at the level of transcription, but also at the level of mRNA translation. Considering these characteristics, it is possible that YB-1 might regulate its own expression at the transla- tional level. The present paper provides evidence that the translational control of YB-1 expression is mediated via the 5¢-UTR of YB-1 mRNA. Global control of translational ef®ciency can be achieved by regulating the phosphorylation state of an array of initiation factors (such as eIF2 and eIF4E). However, control of translational initiation on an individual mRNA is deter- mined primarily by its nucleotide sequence and the secondary structure of the regulatory protein. The 5¢-UTR plays a particularly important role in the regulation of translation initiation via an interaction with RNA binding proteins or by secondary structure formation, which hinders the activity of the translational machinery. Analysis of the 5¢-UTR sequence Figure 8. Effect of mutation of the YB-1 5¢-UTR mRNA element on translation. (A) Schematic representation of the YB-1 5¢-UTR±luciferase of YB-1 (331 bases in length) revealed several important fusion reporter constructs used in this study. pT7-YBM1~3 containing an features that are known to in¯uence translation. internal mutation (mut1-3), designated by the nucleotide at the 5¢ end that Many in vitro and in vivo studies have shown that mRNAs corresponded to the transcription initiation sites (*) (see Fig. 1A). (B and C) with a high likelihood of forming a stable secondary structure The characterization of translation inhibition by each reporter construct was in the 5¢-UTR tend to be inef®ciently translated (34). compared to that of the other constructs. The indicated amounts of GST±YB-1 fusion were added to a rabbit reticulocyte lysate system either However, in both in vitro and in vivo experiments we found initially (B) or after 10 min incubation (C) using 50 ng of the RNA that the 5¢-UTR sequence of YB-1 mRNA, with its high G+C constructs described in (A). The in vitro translation was performed for content, acts as a potent translational enhancer. The full-length 30 min at 30°C and the translation activity of each experiment was meas- 5¢-UTR of YB-1 mRNA increased the level of translation ured. Data are shown as the means 6 SD (error bars) of three independent experiments. activity and cloning a 134 base region of the YB-1 5¢-UTR 620 Nucleic Acids Research, 2004, Vol. 32, No. 2 upstream of the coding initiation sequence was also associated YB-1 has been reported to be involved in the regulation of with a signi®cant increase in translation activity (Fig. 2A and stress-inducible target genes (40,41), suggesting that YB-1 B). This region might therefore be crucial for the translation itself is a stress-activated protein. YB-1 mRNA accumulates activity of the 5¢-UTR of YB-1 mRNA. These data suggest in cells when they are treated with UV irradiation or that elements within the 5¢-UTR of YB-1 mRNA can act as anticancer agents. We previously reported that c-myc and enhancers of mRNA translation. p73 activate YB-1 transcription and may regulate important According to the scanning mechanism postulated for biological processes via their effects on YB-1 gene expression translation initiation, the small (40S) ribosomal subunit enters (42). Additional experiments are needed to con®rm the role of at the 5¢ end of the mRNA and migrates linearly, stopping YB-1 in regulating translation in vivo under various stress when the ®rst AUG codon is reached (35). Nekrasov et al. (31) conditions. It would be of interest to determine how the 5¢- demonstrated that YB-1 protein inhibited the initiation step of UTR of YB-1 mRNA affects the translation of YB-1 in translation, but did not inhibit the elongation step. Rabbit YB- response to extracellular stimuli or environmental stress. 1 displays both RNA melting and annealing activities in a The translational control of gene expression has been dose-dependent manner; a relatively low amount of YB-1 identi®ed as an important regulatory mechanism for many promotes the formation of RNA duplexes, whereas an excess gene products involved in the regulation of proliferation [e.g. of YB-1 causes unwinding of double-stranded forms (36). It is c-mos (43), FGF-2 (44,45), PDGF-B (46), p27 (47) and cdk4 also possible that YB-1 additionally facilitates movement of (48)]. The tumor suppressor p53 has been implicated in the the small ribosomal subunit to the initiation codon complexed translational regulation of both p53 and cdk4 mRNAs (49). Met with initiator tRNA . This may occur up to the initiation Similarly, several distinct components of the translation codon by YB-1 melting the secondary structure in the mRNA apparatus have been shown to be deregulated or overexpressed in human tumors. So, it is of note that the level of the YB-1 5¢-UTR (31). mRNA from the 5¢ furthest transcription initiation start site In this study, we observed that YB-1 inhibited the was signi®cantly higher in HUVEC cells, compared to the translation of all of the transcripts of YB-1 5¢-UTR that other cancer cell lines examined (Fig. 1). The deregulation of lacked sequence speci®city, suggesting that YB-1 reduced the the translational control of YB-1 might therefore also play a translation ef®ciency of 5¢-UTR elements, either at the role in tumor progression. initiation step or by binding directly to the whole RNA. In conclusion, YB-1 protein binds to an RNA containing the These results provided evidence that YB-1 might block the 5¢-UTR of human YB-1 mRNA in vitro and in vivo and ®rst step in mRNA recruitment into translation initiation. exhibits functional activity by inhibiting the translation of a The addition of YB-1 10 min after initiation inhibited YB-1 5¢-UTR±luciferase reporter mRNA through the aid of its translational activity only when the full-length 5¢-UTR of YB- own 5¢-UTR mRNA. We propose a model in which YB-1 1 mRNA was present (Figs 7B and 8C). No repression of the protein autoregulates its own translation by binding to the 5¢- luciferase activity of the deleted or mutant constructs (pT7- YBM1±pT7-YBM3) (with disrupted stem±loop structures) UTR of its own mRNA. Our results support a trans-acting was observed. We also observed that YB-1 binds to the ®rst repressor hypothesis, in which a repressor protein speci®cally half-region of YB-1 5¢ UTR RNA more ef®ciently than does binds to its own 5¢-UTR element, resulting in translational the latter half construct comprising the second half (Fig. 7). repression and the maintenance of constant protein levels. The af®nity of YB-1 for the mutated probes was the same as for the wild-type YB5¢-1 probes (data not shown). These results suggest that ribosome scanning or ongoing translation ACKNOWLEDGEMENTS might be required for repression by YB-1 when the full-length We would like to thank Prof. R.G. Deeley for helpful YB-1 5¢-UTR was used. And when mutated and deleted probe discussions and for critically reading this manuscript. This were used, YB-1 did not affect ribosomal scanning due to work was supported by the Second-Term Comprehensive Ten- alterations in the secondary structure of the YB-1 5¢ UTR. The Year Strategy for Cancer Control from the Ministry of Health precise secondary structures of the YB-1 5¢-UTR remains to and Welfare of Japan and by the Cancer Research Fund from be determined. the Ministry of Education, Culture, Sports, Science and By combining the results presented here with previous data, Technology. we are able to describe the effect of YB-1 on translation. First, YB-1 blocks the initial step in mRNA recruitment to translation initiation, YB-1 then turns off the interaction of REFERENCES the mRNA with eIF4F (31). Second, YB-1 promotes move- ment of the small ribosomal subunit within the complex by 1. Pisarev,A.V., Skabkin,M.A., Thomas,A.A., Merrick,W.C., melting the mRNA 5¢-UTR secondary structure, suggesting Ovchinnikov,L.P. and Shatsky,I.N. 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Nucleic Acids Research – Oxford University Press

Published: Jan 16, 2004

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