©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glyceraldehyde-3-phosphate Dehydrogenase Selectively Binds AU-rich RNA in the NAD-binding Region (Rossmann Fold) (*)

(Received for publication, August 3, 1994; and in revised form, October 10, 1994)

Eszter Nagy (1)(§) William F. C. Rigby (1) (2) (3)(¶)

From the  (1)Departments of Medicine and (2)Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756 and the (3)Veterans Administration Medical Center, White River Junction, Vermont 05009

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A 36-kDa protein that binds AU-rich RNA was purified from human spleen and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH has been previously demonstrated to bind tRNA with high affinity. Competition studies suggested that cytoplasmic GAPDH binds the AU-rich elements (AREs) of lymphokine mRNA 3`-untranslated regions with higher affinity than tRNA. The AUUUA-specific RNA binding activity of GAPDH was inhibited by NAD, NADH, and ATP in a concentration-dependent manner, suggesting that RNA binding of GAPDH might involve the NAD-binding region, or dinucleotide-binding (Rossmann) fold. This hypothesis was supported by experiments that localized RNA binding to the predicted N-terminal 6.8-kDa peptide, known to be involved in the formation of the NAD-binding domain. The direct demonstration of AREspecific binding protein activity localized to the NAD-binding region of GAPDH supports the general concept that enzymes containing this domain may exhibit specific RNA binding activity and play additional roles in nucleic acid metabolism. Finally, cytoplasmic GAPDH was found in the polysomal fraction of T lymphocytes. Thus, the RNA binding specificity of GAPDH as well as its localization within the cell merit its strong consideration as a protein important in the regulation of ARE-dependent mRNA turnover and translation in addition to its well described role in glycolysis.


INTRODUCTION

Post-transcriptional regulation of gene expression has been demonstrated to play a major role in the regulation of cell growth and differentiation in eukaryotic cells (reviewed in (1) ). In T lymphocytes, mRNA turnover represents an important mechanism by which lymphokine production is modulated following activation(2, 3, 4) . The post-transcriptional regulation of lymphokine and proto-oncogene gene expression has resulted in the discovery of highly conserved AU-rich sequences in the 3`-untranslated region that function in cis as destabilizing determinants ((5, 6, 7) ; reviewed in (8) ). These AU-rich elements (AREs) (^1)consist of reiterations of the pentanucleotide AUUUA alone or oligo(U) sequences in an AU-rich context. AREs have been shown to serve as binding sites for cytoplasmic and nuclear proteins that may function as trans-acting factors in regulating ARE-dependent mRNA turnover and translation. A variety of ARE-specific binding proteins (AUBPs) have been described(9, 10, 11, 12, 13, 14, 15, 16, 17, 18) , together with the recent identification of hnRNP A1 as a cytoplasmic AUBP in activated T lymphocytes(19) .

Despite these findings, the molecular and cellular mechanisms by which these AUBPs modulate the turnover of labile mRNAs remain unclear. Recent work correlated a marked increase in the stability of provirally modified IL-2 mRNA with the enhanced binding of hnRNP A1 to AREs in vitro(20) . These findings suggested that hnRNP A1 may serve multiple roles in RNA metabolism in addition to its role in RNA processing, perhaps as a function of its location (nuclear versus cytoplasmic) in the cell(19, 21) . Indeed, many of the proteins important in the regulation of the turnover and translation of mRNA may serve additional and quite dissimilar roles as demonstrated with cellular iron metabolism and the reciprocal regulation of the iron response element-binding protein and aconitase activity(22, 23) . In this regard, the number of enzymes with specific RNA binding activity is steadily growing, including glutamate dehydrogenase(24) , NAD-dependent isocitrate dehydrogenase (25) , thymidylate synthetase(26) , dihydrofolate reductase(27) , catalase(28) , and thiolase(15) . Thus, abundant metabolic enzymes may be involved in or regulate many processes involved in cellular homeostasis independent of the activity that characterized their initial description (reviewed in (29) ).

In an attempt to identify novel AU-rich RNA-binding proteins, a 36-kDa AUBP purified from human spleen was identified by N-terminal microsequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is a key glycolytic enzyme, utilizing NAD as a coenzyme for the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate when assembled as a tetramer of identical 36-kDa subunits. Of interest, numerous non-glycolytic activities have been attributed to GAPDH, including nucleic acid binding(30, 31, 32, 33, 34) , helicase activity(31) , DNA repair(35) , and interaction(s) with actin cytoskeleton (36) and microtubules(37) . Of particular pertinence is the report that the 36-kDa subunit of GAPDH was purified from the nuclei of HeLa cells as a tRNA-binding protein implicated in nuclear export of tRNA(38) . In this study, we characterize the AUBP activity of GAPDH and demonstrate that RNA binding occurs in the Rossmann fold or the NAD-binding region, a structural feature highly conserved among NAD-dependent dehydrogenases(39) . This observation thus permits a rationale for the RNA binding activity recently attributed to several dehydrogenases and other enzymes.


MATERIALS AND METHODS

Reagents

The monoclonal anti-human uracil-DNA glycosylase/GAPDH antibody (40.10.09) was generously provided by Michael Sirover (Temple University School of Medicine, Philadelphia). Plasmid vectors containing tRNA were a gift from Sidney Altman (Yale University School of Medicine, New Haven, CT). Rabbit muscle GAPDH (G 2267), NADH, CAPS, 5,6-dichlorobenzimidazole riboside, and poly(U)-Sepharose beads were purchased from Sigma. NAD, Tween 20, protease inhibitors, and unlabeled nucleotides were from Boehringer Mannheim, while [alpha-P]UTP (3000 Ci/mmol), Hybond-C nitrocellulose membrane, and the ECL chemiluminescence kit for Western blotting were obtained from Amersham Corp. Heparin-Sepharose CL-6B, protein A-Sepharose, and poly(I) were purchased from Pharmacia (Uppsala, Sweden). High and low molecular weight ^14C-labeled and low molecular weight prestained protein standards were obtained from Life Technologies, Inc.

Purification of the 36-kDa Protein

Human spleen obtained at elective splenectomy for hemolytic anemia was minced and then lysed in a Waring blender in homogenization buffer (50 mM HEPES/NaOH, pH 7.5, 25 mM KCl, 5 mM MgCl(2), 250 mM sucrose, 10 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride). Crude lysate was passed through successive layers of cheesecloth to remove unblended material and connective tissue. Nuclei were separated by centrifugation at 1800 times g for 7 min. The supernatant fraction was sequentially precipitated with the addition of 30, 60, and 80% ammonium sulfate. Pellets and the final supernatant fraction (nonprecipitable with 80% (NH(4))(2)SO(4)) were dialyzed against 0.5 times phosphate-buffered saline, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride. Subsequent purification of the 80% (NH(4))(2)SO(4)-precipitated fraction that contained the 36-kDa AUBP was performed using heparin-Sepharose chromatography. The fraction eluted from heparin-Sepharose with 0.25 M NaCl was subjected to poly(U)-Sepharose column chromatography with 0.1 M NaCl in 10 mM sodium phosphate buffer, pH 7.5, and stepwise salt elution. Chromatographic fractions were concentrated and equilibrated in a Centriprep-3 microconcentrator (Amicon, Inc., Beverly, MA) and stored in aliquots in 50 mM NaCl, 10 mM sodium phosphate buffer, pH 7.5, at -70 °C. Protein concentrations were determined using the Micro BCA protein assay reagent (Pierce). RNA binding activity of the fractions was determined by in vitro label transfer assay throughout purification (see below). For microsequence analysis, the 0.25 M NaCl/poly(U) elution fraction was separated by Tris/Tricine-PAGE(40) ; transferred to Problott polyvinylidene difluoride membrane (Applied Biosystems, Inc., Foster City, CA) in 10 mM CAPS, pH 11.0, with 10% methanol; and stained with 0.1% Coomassie Brilliant Blue R-250 in 1% acetic acid, 40% methanol. The 36-kDa band was cut out and analyzed on a Model 476A protein sequencer (Applied Biosystems, Inc.) at the Dartmouth Protein Sequencing Facility.

Cytoplasmic Lysate Preparation

Human peripheral blood mononuclear cells were obtained from healthy volunteers by leukapheresis and isolated by Ficoll-Hypaque density gradient centrifugation. Cells were cultured at 4 times 10^6 cells/ml in RPMI 1640 medium (KC Biologicals, St. Louis, MO) supplemented with 8% heat-inactivated (56 °C, 1 h) fetal bovine serum (Flow Laboratories, Inc.) and 50 µg/ml gentamycin sulfate (U. S. Biochemical Corp.) at 37 °C in a humidified atmosphere of 5% CO(2) in air. Cells were washed twice in ice-cold phosphate-buffered saline or serum-free medium. Preparation of polysomal fractions was according to Brewer and Ross(41) .

Preparation of in Vitro Transcripts

RNA transcripts of high specific activity (>10^8 cpm/µg of RNA) were synthesized from linearized plasmid DNA as a template in the presence of 50 µCi of [alpha-P]UTP, 20 µM UTP, and 4 mM each ATP, GTP, and CTP in 25 µl and incubated at room temperature for 2 h. The complete 3`-UTR of IFN- RNA was prepared by T3 RNA polymerase transcription of BamHI-linearized plasmid pT7/T3alpha19 containing the 665-base pair AluI fragment of the human IFN- gene containing 62 base pairs from the coding region and the full-length 3`-UTR (42) inserted into the SmaI site of the multiple cloning site. The 350-base pair 3`-fragment of the IFN- 3`-UTR containing all the AUUUA sequences was prepared by T3 RNA polymerase transcription of NcoI-linearized plasmid pT7/T3alpha19. This latter shorter IFN- 3`-UTR probe was used in all the experiments, except during the purification and identification of the 36-kDa protein, where the longer 3`-UTR probe was used. The c-myc 3`-UTR probe was prepared by SP6 RNA polymerase transcription of SspI-linearized plasmid pRK5 with the 400-base pair NsiI-AflII human c-myc fragment inserted into the SmaI site of the multiple cloning site(43) . The XhoI fragment of the pXM vector containing the human GM-CSF DNA (provided by the Genetics Institute) was subcloned into the multiple cloning site of the pT7/T3alpha19 plasmid at the BamHI site. The GM-CSF RNA probe was generated by T7 RNA polymerase transcription of this plasmid linearized with EcoRI. The 1, 2, 3, or 4 times AUUUA pentamer-containing probes were generated from a 108-base pair synthetic DNA template containing the T7 promoter (generously provided by James Malter), which was digested with the PstI, BamHI, SacII, or EcoRI restriction enzyme, respectively. The IL-2 3`-UTR RNA probe was prepared by transcription of EcoRI-linearized plasmid pT7/T3alpha19 with the 270-base pair StuI-EcoRI fragment of human IL-2(44) . The Delta2RI probe, which contains four consecutive AUUUA sequences(9) , was prepared by T7 RNA polymerase transcription of EcoRI-linearized plasmid pT7/T3alpha19 with four reiterated AUUUA sequences in the BamHI site of the multiple cloning site. The Delta2H3 RNA probe was transcribed by T3 RNA polymerase using the above plasmid as template, linearized with HindIII. The ptRNA RNA was transcribed by T7 RNA polymerase using AvaI-linearized plasmid pT7/T3alpha19 containing the human ptRNA gene (generously provided by Sidney Altman). The ptRNA(f) RNA probe was prepared by SP6 RNA transcription of the BstNI fragment of plasmid SP64 containing the yeast mitochondrial ptRNA(f) gene(45) . Unlabeled probes were prepared in the presence of 4 mM each ATP, GTP, CTP, and UTP at 30 °C for 2 h.

RNA Binding Assay

RNA probes (4-8 times 10^4 cpm, 0.2-0.8 ng) were incubated with the 0.25 M NaCl/poly(U)-Sepharose chromatographic fraction from human spleen containing the 36-kDa protein/GAPDH or the commercial rabbit muscle GAPDH preparation in 12 mM HEPES, pH 7.9, 15 mM KCl, 0.2 µM dithiothreitol, 0.2 µg/ml yeast tRNA, and 10% glycerol for 10 min at 30 °C. In experiments using radiolabeled tRNA probes and in competition experiments, 0.5-2.5 µg/ml poly(I) was used instead of yeast tRNA to block nonspecific binding. In competition experiments, unlabeled competitors were added simultaneously with the radiolabeled RNA probe. The calculations of molar excess of unlabeled competitors were based on the molecular weights of the different probes relative to that of the radiolabeled probe. UV cross-linking of RNA-protein complexes was performed on ice using Stratalinker 1800 (Stratagene; 5 min, 3000 microwatts/cm^2), followed by RNase digestion (7.5 units of RNase T1 and 15 µg of RNase A for 30 min at 37 °C). Samples were analyzed under reducing and denaturing conditions by 12 or 15% SDS-PAGE according to Laemmli (46) and by autoradiography.

Immunoprecipitation

Purified spleen GAPDH was incubated with P-labeled IFN- 3`-UTR RNA probe, UV-cross-linked, and digested with RNases as described above. Immunoprecipitation of RNA-protein complexes was performed using monoclonal antibody prepared against GAPDH (40.10.09) or control antibody (P3, parent hybridoma supernatant) in buffer IP (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM MgCl(2), 0.5% Triton X-100, 1 µM Pefabloc, 1 µg/ml each leupeptin and pepstatin) and incubation for 1 h on ice. Rabbit anti-mouse whole IgG (Cappel) and protein A-Sepharose beads were incubated for 1 h on ice and then added to RNA-protein-primary antibody complexes and incubated on ice with occasional mixing for 30-45 min. Beads were pelleted by centrifugation; the supernatants (depleted fraction) were collected; and then the beads were washed twice with 1 ml of buffer IP, boiled in Laemmli SDS sample buffer(46) , and analyzed by 12% SDS-PAGE.

Immunoblotting

Proteins were separated by 12% SDS-PAGE and electrotransferred to Hybond-C nitrocellulose membrane in 48 mM Tris, 39 mM glycine buffer, pH 9.1, or in CAPS, pH 11.0, with 15% methanol. The membranes were washed with Tris-buffered saline, 0.075% Tween 20; 5% ECL blocking reagent was used for blocking nonspecific binding. Specific antigen-antibody reaction was detected by chemiluminescence.

Fingerprinting of the GAPDH RNA-binding Site with Staphylococcus aureus V8 Protease

2 µg of rabbit muscle GAPDH was digested with sequencing-grade S. aureus V8 protease in 50 mM NH(4)HCO(3) buffer, pH 7.8, 15 mM KCl, 10% glycerol at different protein/protease ratios for 20 or 240 min at 37 °C, before or after RNA binding in the same buffer at 30 °C, followed by UV cross-linking and RNase digestion as described above. Under these conditions, V8 protease cleaves only at the C-terminal side of glutamic acid residues(47) . Digestion was stopped by boiling the samples in Laemmli SDS sample buffer. The peptides were analyzed on 17% Tris/Tricine gels and visualized by silver nitrate staining (Bio-Rad) and autoradiography.

Northwestern Blotting and Microsequencing

20 µg of rabbit muscle GAPDH was incubated with or without 10 µg of sequencing-grade V8 protease in 50 mM NH(4)HCO(3) buffer, pH 7.8, 15 mM KCl, 10% glycerol for 4 h at 37 °C. Undigested and digested GAPDHs were separated on 17% Tris/Tricine gels and transferred to Hybond-C nitrocellulose or polyvinylidene difluoride membrane in 10 mM CAPS, pH 11, containing 20% methanol. The nitrocellulose membrane was preincubated in 12 mM HEPES, pH 7.9, 15 mM KCl, 0.2 µM dithiothreitol, 0.2 µg/ml yeast tRNA, and 15% glycerol for 10 min at room temperature. RNA binding was performed by adding fresh incubation buffer containing 2 times 10^5 cpm/ml P-labeled IFN- 3`-UTR RNA probe and incubating for 15 min at room temperature with continuous agitation. After washing the blot with binding buffer, it was stained with 0.1% Coomassie Brilliant Blue R-250 in 1% acetic acid, 40% methanol; dried; and subjected to autoradiography. The polyvinylidene difluoride membrane was stained in the same way, and the 6-kDa peptide corresponding to the 6-kDa RNA-binding fragment detected by Northwestern blotting was excised and microsequenced.


RESULTS

Purification of the 36-kDa AUBP and Its Identification as GAPDH

A 36-kDa AUBP was purified from the cytoplasmic fraction of human spleen by following IFN- 3`-UTR RNA binding activity. Sequential ammonium sulfate precipitations substantially enriched the 36-kDa protein in the 80% fraction. This was followed by heparin-Sepharose and poly(U)-Sepharose chromatography, resulting in the presence of a single band on Coomassie Brilliant Blue-stained gels, although additional proteins with lower molecular weights were present upon silver nitrate staining (data not shown). The protein displayed insensitivity to trypsin in a limited proteolysis assay (in contrast to hnRNP A1) (19) and showed no immunological cross-reactivity with human hnRNP A1 (data not shown).

Sequencing of the N-terminal 23 amino acids of the 36-kDa AUBP revealed identity with the N terminus of human GAPDH. Immunoblotting with the 40.10.09 monoclonal antibody raised against human GAPDH reacted with the purified 36-kDa protein, but not with rabbit GAPDH (Fig. 1A). To demonstrate that the RNA binding was mediated by GAPDH, we determined that the anti-human GAPDH monoclonal antibody immunoprecipitated the radiolabeled RNA-protein complex (Fig. 1B). A 25-kDa protein with AUBP activity that was copurified with the 36-kDa protein/GAPDH was not recognized by the anti-GAPDH antibody. Its identity is unknown. Finally, purified 36-kDa AUBP and rabbit muscle GAPDH bound the ARE of the IFN- 3`-UTR with similar activity (Fig. 1C). In other studies, we were able to specifically immunoprecipitate GAPDH complexed to radiolabeled RNA by UV cross-linking from cytoplasmic lysates of phytohemagglutinin-activated human lymphocytes (data not shown), indicating the activity of GAPDH as an RNA-binding protein in these cells.


Figure 1: Identification of the 36-kDa AU-rich RNA-binding protein as glyceraldehyde-3-phosphate dehydrogenase. A, Western blotting. 1.5 µg of protein of the 0.25 M NaCl/poly(U) fraction and 1.5 µg of rabbit muscle GAPDH (Sigma) were analyzed by Western blotting using the anti-uracil-DNA glycosylase/GAPDH antibody (40.10.09). B, immunoprecipitation. 1.5 µg of protein of the 0.25 M NaCl/poly(U) fraction was UV-cross-linked to the [P]UTP-labeled IFN- 3`-UTR, and the RNA-protein complex was immunoprecipitated with control (P3) or specific antibody (40.10.09) as described under ``Materials and Methods.'' The depleted supernatants and the immunoprecipitated complexes were analyzed by SDS-PAGE and autoradiographed. C, binding of rabbit muscle GAPDH to the IFN- 3`-UTR. 0.5 µg of commercial GAPDH or 0.5 µg of the 0.25 M NaCl/poly(U) fraction was assayed for binding to the [P]UTP-labeled IFN- 3`-UTR.



Sequence-specific Binding of the 36-kDa Protein/GAPDH to AU-rich RNA

The 36-kDa protein/GAPDH was purified by measuring IFN- 3`-UTR RNA binding activity. To characterize the specificity of this binding activity, GAPDH was examined for its ability to bind ARE-containing mRNA from other proto-oncogene and cytokine mRNAs (sequences depicted in Fig. 2A). GAPDH bound radiolabeled GM-CSF and the c-myc 3`-UTR to a greater degree than the IFN- 3`-UTR (Fig. 2B). The number and proximity of reiterated AUUUA pentamers necessary for GAPDH binding were examined using radiolabeled RNA probes that contained one, two, three, or four AUUUA reiterations separated by 20 unrelated nucleotides (Fig. 2C). Strong binding of GAPDH to RNA required at least three reiterations of AUUUA, although a single AUUUA motif generated detectable signal. Thus, GAPDH binds several AREs that differ considerably in both their sequence as well as the proximity of AUUUA pentamers. This is further supported by the finding of equivalent binding between the sense and antisense IFN- 3`-UTR probes, the latter containing two AUUUA and one AUUUUA motif (data not shown).


Figure 2: Specificity of binding of GAPDH to AU-rich RNA. A, schematic representation of the sequences of the RNA probes used in RNA binding assays. B, binding of GAPDH to different AU-rich lymphokine and cytokine RNAs. [P]UTP-labeled IFN- 3`-UTR, GM-CSF, and c-myc 3`-UTR RNA probes were incubated with 0.75 µg of splenic GAPDH (0.25 M NaCl/poly(U) fraction), UV-cross-linked, and analyzed by SDS-PAGE. C, binding of GAPDH to separated AUUUA motifs. 0.2 µg of splenic GAPDH was incubated with P-labeled RNA probes containing one, two, three, or four AUUUA pentamers as described under ``Materials and Methods.'' (Equal molars amount of radioactive probes were added, corrected for the number of U nucleotides.) D, competition of lymphokine-cytokine mRNA 3`-UTRs with binding of GAPDH to the IFN- 3`-UTR. 0.2 µg of splenic GAPDH was assayed for binding to the [P]UTP-labeled IFN- 3`-UTR in the presence of the indicated unlabeled RNA sequences. 2.5 µg/ml poly(I) was used to block nonspecific RNA binding.



The specificity of the GAPDH-ARE interaction was further studied in competition studies (Fig. 2D). The AUUUA sequence specificity was confirmed by demonstrating that the unlabeled in vitro transcribed IFN- and IL-2 3`-UTRs as well as a ribo-oligonucleotide containing the sequence 5`-AUUUAUUUAUUUAUUUA-3` (Delta2R1 probe) effectively competed for binding, while the addition of the nonspecific inhibitor Delta2H3 (antisense Delta2R1) had little or no effect on complex formation. In other studies, GAPDH binding to the IFN- and c-myc 3`-UTRs was found to greatly exceed that found in a ribo-oligonucleotide with five continuous reiterated AUCUA sequences, further indicating specificity for oligouridine or AUUUA sequences (data not shown). Further information on the specificity of GAPDH binding to RNA was obtained with the testing of the ability of unlabeled ribohomopolymers to compete for binding to the P-labeled IFN- 3`-UTR. Poly(U) was superior to either poly(C) or poly(A) at reducing binding, with poly(A) demonstrating an intermediate level of inhibition (Fig. 3).


Figure 3: Competition of binding with ribohomopolymers. A, 0.2 µg of rabbit muscle GAPDH was assayed for binding to the [P]UTP-labeled IFN- 3`-UTR in the absence or presence of the indicated amounts of poly(U), poly(C), or poly(A) RNA. B, the RNA-protein complexes were analyzed as described under ``Materials and Methods,'' and the results of densitometric reading of the autoradiogram are expressed as percent binding to GAPDH in the presence of the indicated competitors relative to control.



GAPDH was recently identified as a nuclear tRNA-binding protein(38) . The binding of the two types of RNA ligands (tRNA and the ARE-containing 3`-UTR) by GAPDH was examined with selected tRNAs, ptRNA (human precursor serine tRNA) and ptRNA(f) (yeast mitochondrial precursor methionine tRNA). GAPDH bound the IFN- 3`-UTR more strongly than the tRNA probes, exhibiting a rank order of binding of IFN- 3`-UTR > ptRNA(f) > ptRNA (Fig. 4A). Consistent with this observation, both unlabeled in vitro transcribed ptRNA(f) and the yeast tRNA mixture were less efficient than the unlabeled IFN- 3`-UTR at competing with GAPDH binding to the radiolabeled IFN- 3`-UTR (Fig. 4B).


Figure 4: Comparison of binding of GAPDH to the ARE-containing 3`-UTR and tRNA. A, binding of GAPDH to the radiolabeled IFN- 3`-UTR, ptRNA(f), and ptRNA. 0.2 µg of rabbit muscle GAPDH was incubated with the indicated [P]UTP-labeled RNA probes in the presence of 0.5 µg/ml poly(I). (Equal molar amounts of radioactive probes were added, corrected for the number of U nucleotides of the different probes.) B, competition of binding of GAPDH to the IFN- 3`-UTR with tRNAs. 0.2 µg of splenic GAPDH was assayed for binding to the [P]UTP-labeled IFN- 3`-UTR in the presence or absence of the indicated molar excess of the unlabeled in vitro transcribed IFN- 3`-UTR and ptRNA(f) and yeast tRNA (Sigma). 0.5 µg/ml poly(I) was used to block nonspecific RNA binding.



Regulation of the RNA Binding Activity of GAPDH

Previous studies have demonstrated that the binding activity of both the iron response element-binding protein and AUBP is modulated by the redox state of the proteins(48, 49) . These observations prompted us to study the possible role of redox changes in the AUBP activity of GAPDH. Incubation of GAPDH with the reducing agent 2-mercaptoethanol enhanced the binding of both lymphokine 3`-UTR and tRNA probes (Fig. 5A). In contrast, treatment with the oxidizing agent diamide (10 mM) markedly decreased the binding of GAPDH to the [P]UTP-labeled IFN- 3`-UTR (Fig. 5B). This effect of diamide could be reversed by subsequent treatment with 2-mercaptoethanol. These data are very similar to those observed with the iron response element-binding protein (48) and an AUBP described previously(49) , raising the possibility that SH groups in GAPDH may also function intracellularly as a ``sulfhydryl switch'' by which its RNA binding activity is regulated.


Figure 5: RNA binding activity of GAPDH is regulated by redox changes. A, effect of 2-mercaptoethanol (2-me) on GAPDH binding to both the ARE and tRNA. 0.25 µg of splenic GAPDH was preincubated for 5 min at room temperature in the absence or presence of 1% 2-mercaptoethanol prior to incubation with the radiolabeled IFN- 3`-UTR, ptRNA(f), and ptRNA (as described in the legend to Fig. 4A). RNA-protein complexes were analyzed by nonreducing SDS-PAGE. B, effect of diamide on the AUBP activity of GAPDH. 0.2 µg of splenic GAPDH was preincubated with 10 mM diamide for 10 min at room temperature, and then the indicated amount of 2-mercaptoethanol was added for 5 min at room temperature. Radiolabeled GAPDHbulletIFN- 3`-UTR RNA complexes were analyzed as described under ``Materials and Methods.''



NAD and NADH are necessary for the glycolytic function of GAPDH. It has been reported that incubation of GAPDH with NAD reduces its RNA binding activity(30, 38) . Experiments were therefore undertaken to determine if the same was true of AUBP binding activity. Increasing concentrations of NAD and NADH decreased GAPDH binding of both [P]UTP-labeled IFN- 3`-UTR (Fig. 6A, upper panel) and ptRNA(f) (lower panel) RNA probes. These data indicate that NAD and NADH may regulate RNA binding by GAPDH to both types of RNAs. The specificity of this inhibitory effect on GAPDH-RNA interactions was supported by the lack of effect of these coenzymes on AUBP activity on the serendipitously copurified 25-kDa protein.


Figure 6: Inhibition of RNA binding of GAPDH by coenzymes NAD and NADH and by ATP. A, 0.4 µg of splenic GAPDH was preincubated for 5 min at room temperature with the indicated concentrations of NAD and NADH prior to binding to the [P]UTP-labeled IFN- 3`-UTR (upper panel) or ptRNA(f) (lower panel). B, 0.25 µg of splenic GAPDH was assayed for binding to the [P]UTP-labeled IFN- 3`-UTR in the presence of the indicated concentrations of ATP or UTP. RNA-protein complexes were analyzed as described under ``Materials and Methods.''



It has been reported that GAPDH is capable of autophosphorylation in vitro(50) . This prompted us to study whether the AUBP activity of GAPDH can be modulated by autophosphorylation. GAPDH was preincubated with ATP and/or Mg under conditions to promote autophosphorylation (50) and then analyzed for binding to the [P]UTP-labeled IFN- 3`-UTR probe. Preincubation with 1 mM ATP alone or ATP + Mg reduced the binding of GAPDH to AU-rich RNA dramatically (data not shown). This effect was equally apparent whether the incubation was carried out at 0 or 30 °C, further suggesting that ATP binding to GAPDH, and not phosphorylation, altered AUBP activity. To examine the specificity of this effect, ATP or UTP was added to the binding solution at increasing concentrations without preincubation or Mg. ATP progressively decreased GAPDH binding to RNA, with half-maximal inhibition at 50 µM, while a 20-fold higher concentration of UTP was needed to mediate comparable inhibition (Fig. 6B).

Localization of the RNA-binding Site on GAPDH

Our data demonstrate that the in vitro binding of GAPDH to the ARE-containing 3`-UTR and tRNA is decreased by the addition of NAD, NADH, and ATP. Since all of these agents can bind to the NAD-binding domain(51) , we directly addressed if the site of RNA binding by GAPDH is in the Rossmann fold, a structural domain highly conserved among dehydrogenases(39) . GAPDH was subjected to proteolysis by S. aureus V8 protease under nondenaturing conditions. For these experiments, the commercially available rabbit muscle GAPDH preparation was used as it demonstrated equivalent RNA binding specificity to human splenic GAPDH, unsurprising given the high level (94%) of amino acid identity. The expected fragments of GAPDH upon complete digestion with V8 protease are depicted in the peptide map shown in Fig. 7A. GAPDH was digested with V8 protease at different protease/protein ratios for various times (20 or 240 min), both prior to (Fig. 7B) and after (Fig. 7C) incubation with the radiolabeled IFN- 3`-UTR. Increasing V8 protease concentrations were associated with the progressive disappearance of the 36-kDa AUBP, which represents the intact subunit of GAPDH, and with the eventual generation of five peptides with AUBP activity ranging from 6 to 25 kDa (Fig. 7B, 20-min incubation at 1:30 and 1:5 ratios). With further digestion, the larger peptides (25, 20, and 16 kDa) with AUBP activity disappeared (Fig. 7B, 240-min incubation at a 1:5 ratio), while the smaller ones (10 and 6 kDa) increased. The progressive appearance and stability of the 6-kDa fragment to further proteolysis suggest that this peptide might represent the N-terminal fragment of rabbit GAPDH with a calculated size of 6.8 kDa. Thus, the RNA binding activity of GAPDH appears to be localized to a peptide that is part of the NAD-binding domain, or Rossmann fold(39) .


Figure 7: Fingerprinting of the RNA-binding domain of GAPDH. Shown is schematic representation of the predicted V8 protease peptide map of GAPDH (A). The subunit of GAPDH consists of two domains. The first half of the subunit is the NAD-binding domain, and the second half is for substrate binding, specificity, and catalysis. Cysteine 147 (in rabbit muscle GAPDH) occurs at the junction between the two domains in the center of the subunit. Digestion of 2 µg of rabbit muscle GAPDH with S. aureus V8 protease at the indicated protein/protease ratios was performed before (B) or after (C) binding to the radiolabeled IFN- 3`-UTR for the indicated lengths of time as described under ``Material and Methods.'' Following UV cross-linking and RNase digestion, the proteins and peptides were analyzed by 17% Tris/Tricine-PAGE.



It is worth noting that the generation of peptide fragments by V8 protease digestion was influenced by GAPDHbulletRNA complex formation (Fig. 7C). V8 protease digestion after RNA binding (but prior to UV cross-linking) inhibited the generation of the 6-kDa peptide relative to that observed when V8 protease digestion was performed prior to RNA binding (Fig. 7, B and C, digestion, 1:5 ratio). This effect was accompanied by a corresponding change in the rate of loss of the 10-kDa peptide with AUBP activity, suggesting that RNA binding has made it more resistant to proteolytic attack by V8 protease. One interpretation of this observation is that access of the protease is blocked by RNA binding, thereby localizing the RNA-binding domain of GAPDH to the C-terminal end of the predicted N-terminal 6.8-kDa peptide (Glu-61). The selectivity of the RNA binding activity of the five observed fragments of GAPDH is underscored by the complex digestion pattern demonstrated by silver staining, which reveals at least 15-20 peptides ranging from 3 to 33 kDa (data not shown).

Identification of the observed 6-kDa RNA-binding peptide as the N terminus of GAPDH cannot be made solely on the basis of the predicted size of a proteolytic fragment. Therefore, we determined the amino acid sequence of the observed 6-kDa RNA-binding peptide. GAPDH was digested with V8 protease, and the digestion mixture was separated by electrophoresis and transferred to nitrocellulose membrane. RNA binding performed in situ on the membrane-attached peptides (Northwestern blotting) revealed an 6-kDa fragment (Fig. 8A). Microsequence analysis of the excised 6-kDa peptide identified by RNA binding and Coomassie Brilliant Blue staining (Fig. 8) yielded an amino acid sequence that is 100% identical to that of the N terminus of rabbit muscle GAPDH. These experiments unambiguously localize the RNA-binding domain of GAPDH to its NAD-binding region as well as demonstrate RNA binding by GAPDH in the absence of UV cross-linking.


Figure 8: Identification of the IFN- 3`-UTR-binding peptide fragment of GAPDH as the NAD-binding region (Rossmann fold). Rabbit muscle GAPDH was digested with V8 protease and then transferred to nitrocellulose membrane. RNA-binding proteins were detected by Northwestern blotting (as described under ``Materials and Methods'') (A); total proteins were visualized by Coomassie Brilliant Blue staining (B). The 6-kDa peptide (indicated by the arrows) was excised from the membrane for microsequencing.



Intracellular Localization of GAPDH in T Lymphocytes

Given the identification of GAPDH in this report as a cytoplasmic protein with mRNA binding activity, we examined its subcellular location in the cytoplasm of resting and activated T lymphocytes. Following cell fractionation(41) , polysomal fractions were analyzed by immunoblotting (Fig. 9). In three separate experiments, GAPDH was found in the polysomal fraction of human T cells. Interestingly, polysomal levels of GAPDH appeared to be increased by the transcriptional inhibitor 5,6-dichlorobenzimidazole riboside, similar to data reported for other AUBPs(18, 19) . Thus, the potential role of cytoplasmic GAPDH as a functionally relevant AUBP is strengthened by demonstrating both its polysomal location as well as a common pattern of modulation by RNA polymerase II inhibition relevant to other AUBPs.


Figure 9: Polysomal location of GAPDH and its modulation by transcriptional inhibition. Proteins of the polysomal fractions of 7 times 10^6 peripheral blood mononuclear cells (1 µg) were separated by 12% SDS-PAGE and analyzed by immunoblotting with the anti-GAPDH antibody (40.10.09) as described under ``Materials and Methods.'' Lane1, resting human lymphocytes; lane2, human lymphocytes activated with phytohemagglutinin for 16 h; lane3, phytohemagglutinin-activated human lymphocytes treated with 5,6-dichlorobenzimidazole riboside (100 µM) for 1 h.




DISCUSSION

The presence of the ARE in the 3`-UTR of labile cytokine, lymphokine, and proto-oncogene mRNAs has been shown to be important in the regulation of both mRNA stability (5, 6, 7, 8) and translation(52, 53, 54, 55) . The ability of specific AUBPs to interact with these sequences has been correlated with changes in both nuclear and cytoplasmic mRNA stability (11, 20) . In this paper, we report the identification of a 36-kDa AUBP purified from human spleen as glyceraldehyde-3-phosphate dehydrogenase based on its N-terminal amino acid sequence, immunoprecipitation of GAPDHbulletIFN- 3`-UTR complexes with the anti-GAPDH monoclonal antibody, and the demonstration that rabbit muscle GAPDH shares similar RNA binding activity.

Here we demonstrate that GAPDH binds to AU-rich RNA sequences present in the 3`-untranslated region of IFN-, c-myc, GM-CSF, and IL-2 mRNAs. The observation that GAPDH formed complexes with the 3`-UTR of c-myc very efficiently is intriguing because of the reported dysregulation of c-myc expression in Bloom's syndrome(56) , a condition in which structural alterations in GAPDH have been described(57) . GAPDH was able to bind RNA probes with discontinuous AUUUA pentamers, although three reiterations were necessary for optimalbinding. This is in contrast to observations with hnRNP A1, where little or no binding was observed. (^2)

Of relevance to this finding is the previous report that GAPDH is a tRNA-binding protein implicated in nuclear export of tRNA(38) . Competition studies indicate that both human splenic and rabbit muscle GAPDHs bind the 3`-UTR of IFN- with somewhat higher affinity than two selected tRNA probes. Although these assays do not permit the affinity of GAPDH for the 3`-UTR of IFN- to be measured, previous work demonstrated that GAPDH bound tRNA with a high affinity (K(A) 1.8 times 10M for tRNA)(38) . Based on these data, it would seem reasonable to infer that the GAPDH-ARE binding was also a high affinity interaction. In addition, ptRNA and the ptRNA(f) used in this study contain oligo(U) sequences (CUUUUUUUA and AUUUUA, respectively). In this regard, it is of interest that combinatorial selection studies have demonstrated that the HELN-1 protein, a member of the AUBP family, recognizes oligo(U) sequences (3, 4, or even 5 U nucleotides) flanked by other nucleotides than A(14) . The stronger binding to lymphokine 3`-UTRs relative to tRNAs may therefore have been the result of higher avidity given the presence of reiterated AU-rich sequences that could serve as multiple binding sites for GAPDH, which exists as monomers, dimers, and tetramers in solution. Unfortunately, measurement of RNA binding affinity under native conditions in the absence of UV cross-linking was not successful because of the aggregation of GAPDH in the presence of RNA. However, these studies do permit the identification of AUUUA pentamers and U-rich sequences as sites at which GAPDH can specifically interact with RNA with high affinity. This is in contrast to studies of tRNA binding by GAPDH, where the nature of the sequence recognized by GAPDH was not established(38) .

It is worth noting that multiple AUBPs between 32 and 40 kDa have been described in cytoplasmic and nuclear extracts(9, 10, 11, 13, 17, 18) . Based on similar binding characteristics, one of these RNA-binding proteins may represent GAPDH. We have previously described a cytoplasmic 36-kDa AUBP activity in activated T lymphocytes that consisted of trypsin-sensitive (hnRNP A1) and trypsin-resistant components(19) . Given their relatively comparable, but not identical AUBP activity (see above), and their similar size, it may be that the trypsin-resistant component of the 36-kDa AUBP represented GAPDH, instead of a closely related molecule or isoform of hnRNP A1(58) .

The demonstration that NAD, NADH, and ATP were able to diminish the specific AUBP activity of GAPDH suggested that the dinucleotide-binding (Rossmann) fold of GAPDH might serve as an RNA-binding domain. This was further supported by the finding that inhibition by UTP occurred at a 20-fold higher concentration, consistent with the lower binding affinity of this compound for the NAD-binding region of GAPDH(59) . To date, we are unaware of direct evidence that identifies the Rossmann fold as an RNA-binding domain. Through V8 protease-mediated proteolysis, Northwestern blotting, and microsequencing, we have demonstrated that the AUBP activity of GAPDH was exhibited by the predicted N-terminal 6.8-kDa peptide. As RNA binding of GAPDH retarded generation of this predicted N-terminal 6.8-kDa peptide, it is likely that glutamate 61 was masked from proteolytic attack by the GAPDHbulletRNA complex. Thus, it can be inferred that a component of the RNA-binding domain extends beyond the predicted N-terminal 6.8-kDa peptide. This observation would be consistent with the structure of the NAD-binding domain, which is made up of two roughly identical mononucleotide-binding sites in the NAD-dependent dehydrogenases(39) . In GAPDH, one of the mononucleotide-binding areas is in the predicted 6.8-kDa V8 peptide fragment, with the other residing in the C-terminal half of the dinucleotide-binding fold. Hydrophobic interactions have been shown to be important in coenzyme binding in the NAD-binding domain (39) . Consistent with these findings is our observation that the AUBP activity of GAPDH is unaffected over a wide pH range (from 3.5 to 9.7) (data not shown), suggesting that specific ARE binding by GAPDH is not a consequence of its overall cationic nature (pI 8.1-8.7) or ionic interactions. This insensitivity to pH changes suggests the importance of hydrophobic interactions in RNA binding by GAPDH.

Concurrent with these studies, other members of the family of dehydrogenases (lactate dehydrogenase, yeast mitochondrial NAD-dependent isocitrate dehydrogenase, and glutamate dehydrogenase) and NAD(P)/NAD(P)H-binding enzymes (catalase and dihydrofolate reductase) have been demonstrated to have sequence-specific RNA binding capacity (reviewed in (29) ). Yeast isocitrate dehydrogenase binds the AU-rich 5`-untranslated region of all major yeast mitochondrial mRNAs(25) , while glutamate dehydrogenase has a specific RNA binding activity for cytochrome c oxidase mRNA(24) . Given the absence of RNA-binding domains with characteristic ribonucleoprotein consensus sequences (reviewed in (60) and (61) ) and the similarity in three-dimensional structure of the dinucleotide-binding regions of different dehydrogenases(39, 62) , our data suggest that the Rossmann fold is capable of serving as an RNA-binding domain. The strong evolutionary conservation of the Rossmann fold and its presence in kinases, tRNA synthetases, and NAD(P)- and FAD-dependent dehydrogenases (39, 63) may therefore be relevant to the ability of these proteins to regulate RNA metabolism. Characterization of the NAD-binding region as an RNA-binding domain may therefore have broad implications for both mRNA metabolism and translation, as evidenced by the reciprocal identification of GAPDH as a tRNA-binding protein (38) and some tRNA synthetases as mRNA-binding proteins(64) .

In human T lymphocytes, GAPDH can be localized to the polysomes, supporting the notion that GAPDH binds RNA in vivo. Furthermore, polysomal levels of GAPDH are increased by transcriptional inhibition, similar to data reported for other AUBPs(18, 19) . Thus, the potential role of cytoplasmic GAPDH as a functionally relevant AUBP is strengthened by demonstrating both its polysomal location as well as a common pattern of modulation by RNA polymerase II inhibition. Based on this finding, the local concentration of NAD, NADH, and ATP in different subcellular compartments (polysomes, cytosol, and nucleus) may regulate GAPDH activity. Our data suggest that the Rossmann fold of GAPDH is reciprocally regulated between its RNA binding (inactive in glycolysis) and NAD binding (active in glycolysis) states in vivo. This mechanism would favor glycolytic activity of GAPDH in the cytosol (where the concentration of NAD and ATP is high) by inhibiting RNA binding. Conversely, GAPDH may bind RNA in the polysomal and nuclear microenvironments due to lower concentrations of NAD, NADH, and free ATP. This interpretation is supported by the finding that NAD and NADH blocked IFN- binding to GAPDH at concentrations (1-10 µM) at which they have been reported to associate with its Rossmann fold(65) .

Alternatively, post-translational modifications (redox or otherwise) might influence the intracellular location of GAPDH as well as differentially regulate its RNA or NAD binding activity. In this regard, it is worth noting that purified splenic GAPDH consisted of a single isoform on two-dimensionalPAGE, in contrast to what has been demonstrated for the multiple isoforms of total cellular GAPDH(30, 31) . Thus, isoforms of GAPDH may differ considerably in their affinity for specific RNA ligands. Indeed, Karpel and Burchard (31) reported that only the most basic isoform of yeast GAPDH possesses poly(U) binding capacity and helix destabilizing activity in vitro.

GAPDH has been shown to possess a wide range of biological activities in addition to its well characterized role as a specific dehydrogenase essential in glycolysis. Our studies potentially extend these activities into mRNA metabolism. In this regard, it is intriguing that hnRNP A-type proteins have been shown to shuttle between the nucleus and cytoplasm and are implicated in mRNA export(66, 67) . Given the relatively reciprocal distribution of GAPDH and hnRNP A1 between the nucleus and cytoplasm, their demonstrated AUBP activity, and the elevation of polysomal GAPDH levels with RNA polymerase II inhibition, GAPDH may play a similar role not only in nuclear tRNA export(38) , but also in the export of AU-rich mRNAs, as reported for other AUBPs(19, 68) . In addition to this potential role in nucleocytoplasmic export of tRNA and mRNA, the polysomal location of GAPDH and its RNA binding specificity suggest a role in ARE-dependent mRNA turnover and translation. Given the ability of GAPDH to decrease the melting point of RNA(31) , GAPDH may be important for RNA unwinding during translation or accessibility of the 3`-UTR to endoribonuclease attack and may therefore influence the translatability or stability of mRNA through its binding to the ARE. In contrast to GAPDH, hnRNP A1 has been shown to have annealing activity(69) . As the ability of hnRNP A1 to bind the ARE in vitro correlates with increased mRNA stability in vivo(20) , these data suggest the intriguing possibility that GAPDH and hnRNP A1 may compete for similar, but not identical, RNA ligands in vivo, with profoundly different consequences in terms of mRNA stability and translation.

In conclusion, identification of the Rossmann fold of GAPDH as an RNA-binding site provides new insights into the regulation of this multifunctional protein. Moreover, this finding may be relevant to the RNA binding activity that has been recently described in other dehydrogenases, given the conservation of the Rossmann fold among these enzymes. The RNA binding specificity of GAPDH as well as its polysomal localization not only prompt consideration of this protein as a regulator of ARE-dependent mRNA turnover and translation, but also extend the conceptual framework of cytoplasmic mRNA metabolism to include proteins with previously defined roles in glycolysis.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1 AI2434 and KO4 AI00910 and by research funds from the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Clinical Chemistry, University Medical School, Pécs H-7643, Hungary.

To whom correspondence should be addressed: Section of Connective Tissue Diseases, Dept. of Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756. Tel: 603-650-7700; Fax: 603-650-6223.

(^1)
The abbreviations used are: AREs, AU-rich elements; AUBPs, ARE-specific binding proteins; hnRNP, heterogeneous nuclear ribonucleoprotein; IL-2, interleukin-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAPS, 3-(cyclohexylamino)propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; UTR, untranslated region; IFN-, interferon-; GM-CSF, granulocyte-macrophage colony-stimulating factor; ptRNA, precursor tRNA.

(^2)
B. J. Hamilton and W. F. C. Rigby, unpublished observation.


ACKNOWLEDGEMENTS

We gratefully acknowledge Michael Sirover for provision of antibodies against human uracil-DNA glycosylase/GAPDH as well as James Malter and Sidney Altman for the mRNA and tRNA constructs, respectively. We also thank JoNell Hamilton for the preparation of the IL-2 3`-UTR and GM-CSF constructs, Bradley Arrick for that of the c-myc 3`-UTR construct, and Mary Waugh for the preparation of the polysomal fractions. We thank Tamás Henics, Jacqueline Sinclair, and Peter Sinclair for critical reading of the manuscript.


REFERENCES

  1. Brawerman, G. (1993) in Control of Messenger RNA Stability (Belasco, J. G., and Brawerman, G., eds) pp. 149-159, Academic Press, New York
  2. Taniguchi, T. (1988) Annu. Rev. Immunol. 6, 439-464 [CrossRef][Medline] [Order article via Infotrieve]
  3. Shaw, J., Meerovitch, K., Bleackley, R. C., and Paetkau, V. (1988) J. Immunol. 140, 2243-2248 [Abstract/Free Full Text]
  4. Bickel, M., Cohen, R. B., and Pluznik, D. H. (1990) J. Immunol. 145, 840-845 [Abstract/Free Full Text]
  5. Shaw, G., and Kamen, R. (1986) Cell 46, 659-667 [Medline] [Order article via Infotrieve]
  6. Jones, T. R., and Cole, M. D. (1987) Mol. Cell. Biol. 7, 4513-4521 [Medline] [Order article via Infotrieve]
  7. Shyu, A., Greenberg, M. E., and Belasco, J. G. (1989) Genes & Dev. 3, 60-72
  8. Greenberg, M. E., and Belasco, J. G. (1993) in Control of Messenger RNA Stability (Belasco, J. G., and Brawerman, G., eds) pp. 199-218, Academic Press, New York
  9. Malter, J. S. (1989) Science 246, 664-666 [Medline] [Order article via Infotrieve]
  10. Bohjanen, P. R., Petryniak, B., June, C. H., Thompson, C. B., and Lindsten, T. (1991) Mol. Cell. Biol. 11, 3288-3295 [Medline] [Order article via Infotrieve]
  11. Vakalopoulou, E., Schaack, J., and Shenk, T. (1991) Mol. Cell. Biol. 11, 3355-3364 [Medline] [Order article via Infotrieve]
  12. Brewer, G. (1991) Mol. Cell. Biol. 11, 2460-2466 [Medline] [Order article via Infotrieve]
  13. Bickel, M., Iwai, Y., Pluznik, D. H., and Cohen, R. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10001-10005 [Abstract]
  14. Levine, T. D., Gao, F., King, P. H., Andrews, L. G., and Keene, J. D. (1993) Mol. Cell. Biol. 13, 3494-3504 [Abstract]
  15. Nanbu, R., Kubo, T., Hashimoto, T., and Natori, S. (1993) J. Biochem. (Tokyo) 114, 432-437 [Abstract]
  16. Zhang, W., Wagner, B. J., Ehrenman, K., Schaefer, A. W., DeMaria, C. T., Crater, D., DeHaven, K., Long, L., and Brewer, G. (1993) Mol. Cell. Biol. 13, 7652-7665 [Abstract]
  17. Huang, L.-Y., Tholanikunnel, B. G., Vakalopoulou, E., and Malbon, C. C. (1993) J. Biol. Chem. 268, 25769-25775 [Abstract/Free Full Text]
  18. Katz, D. A., Theodorakis, N. G., Cleveland, D. W., Lindsten, T., and Thompson, C. B. (1994) Nucleic Acids Res. 22, 238-246 [Abstract]
  19. Hamilton, J. B., Nagy, E., Malter, J. S., Arrick, B. A., and Rigby, W. F. C. (1993) J. Biol. Chem. 268, 8881-8887 [Abstract/Free Full Text]
  20. Henics, T., Sanfridson, A., Hamilton, B. J., Nagy, E., and Rigby, W. F. C. (1994) J. Biol. Chem. 269, 5377-5383 [Abstract/Free Full Text]
  21. Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993) Annu. Rev. Biochem. 62, 289-321 [CrossRef][Medline] [Order article via Infotrieve]
  22. Kaptain, S., Downey, W. E., Tang, C., Philpott, C., Haile, D., Orloff, D. G., Harford, J. B., Rouault, T. A., and Klausner, R. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10109-10113 [Abstract]
  23. Klausner, R. D., Rouault, T. A., and Harford, J. B. (1993) Cell 72, 19-28 [Medline] [Order article via Infotrieve]
  24. Preiss, T., Hall, A. G., and Lightowlers, R. N. (1993) J. Biol. Chem. 268, 24523-24526 [Abstract/Free Full Text]
  25. Elzinga, S. D. J., Bednarz, A. L., von Oosterum, K., Dekker, P. J. T., and Grivell, L. A. (1993) Nucleic Acids Res. 21, 5328-5331 [Abstract]
  26. Chu, E., Voeller, D., Koeller, D. M., Drake, J. C., Takimoto, C. H., Maley, G. F., Maley, F., and Allegra, C. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 517-521 [Abstract]
  27. Chu, E., Takimoto, C. H., Voeller, D., Grem, J. L., and Allegra, C. J. (1993) Biochemistry 32, 4756-4760 [Medline] [Order article via Infotrieve]
  28. Clerch, L. B., Wagner, A., and Massaro, D. (1993) FASEB J. 7, A1233 (abstr.) [Abstract/Free Full Text]
  29. Hentze, M. W. (1994) Trends Biochem. Sci. 19, 101-103 [CrossRef][Medline] [Order article via Infotrieve]
  30. Perucho, M., Salas, J., and Salas, M. L. (1977) Eur. J. Biochem. 81, 557-562 [Abstract]
  31. Karpel, R. L., and Burchard, A. C. (1981) Biochim. Biophys. Acta 654, 256-267 [Medline] [Order article via Infotrieve]
  32. Ryazanov, A. G. (1985) FEBS Lett. 192, 131-134 [CrossRef][Medline] [Order article via Infotrieve]
  33. Grosse, F., Nasheuer, H., Scholtissek, S., and Schomburg, U. (1986) Eur. J. Biochem. 160, 459-467 [Abstract]
  34. Morgenegg, G., Winkler, G. C., Hübscher, U., Heizmann, C. W., Mous, J., and Kuenzle, C. C. (1986) J. Neurochem. 47, 54-62 [Medline] [Order article via Infotrieve]
  35. Meyer-Siegler, K., Mauro, D. J., Seal, G., Wurzer, J., DeRiel, J. K., and Sirover, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8460-8464 [Abstract]
  36. Méjean, C., Pons, F., Benyamin, Y., and Roustan, C. (1989) Biochem. J. 264, 671-677 [Medline] [Order article via Infotrieve]
  37. Huitorel, P., and Pantaloni, D. (1985) Eur. J. Biochem. 150, 265-269 [Abstract]
  38. Singh, R., and Green, M. R. (1993) Science 259, 365-368 [Medline] [Order article via Infotrieve]
  39. Rossmann, R. G., Liljas, A., Br ä nd é n, C. I., and Banaszak, L. J. (1975) in The Enzymes (Boyer, P. D., ed) pp. 62-102, Academic Press, New York, and references therein
  40. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  41. Brewer, G., and Ross, J. (1990) Methods Enzymol. 181, 202-209 [Medline] [Order article via Infotrieve]
  42. Gray, P. W., Leung, D. W., Pennica, D., Yelverton, E., Najarian, R., Simonsen, C. C., Derynck, R., Sherwood, P. J., Wallace, D. M., Berger, S. L., Levinson, A. D., and Goeddel, D. V. (1982) Nature 295, 503-508 [Medline] [Order article via Infotrieve]
  43. Alitalo, K., Schwab, M., Lin, C. C., Varmus, H. E., and Bishop, J. M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1707-1711 [Abstract]
  44. Holbrook, N. J., Smith, K. A., Fornace, A. J., Comeau, C. M., Wiskocil, R. L., and Crabtree, G. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1634-1638 [Abstract]
  45. Morales, M. J., Wise, C. A., Hollingsworth, M. J., and Martin, N. C. (1989) Nucleic Acids Res. 17, 6865-6881 [Abstract]
  46. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  47. Houmard, J., and Drapeau, G. R. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 3506-3509 [Abstract]
  48. Hentze, M. W., Rouault, T. A., Harford, J. B., and Klausner, R. D. (1989) Science 244, 357-359 [Medline] [Order article via Infotrieve]
  49. Malter, J. S., and Hong, Y. (1991) J. Biol. Chem. 266, 3167-3171 [Abstract/Free Full Text]
  50. Kawamoto, R. M., and Caswell, A. H. (1986) Biochemistry 25, 656-661
  51. Harris, J. I., and Waters, M. (1976) in The Enzymes (Boyer, P. D., ed) Vol. XIII, pp. 1-49, Academic Press, New York
  52. Kruys, V., Marinx, O., Shaw, G., Deschamps, J., and Huez, G. (1989) Science 245, 852-855 [Medline] [Order article via Infotrieve]
  53. Savant-Bhonsale, S., and Cleveland, D. W. (1992) Genes & Dev. 6, 1927-1939
  54. Aharon, T., and Schneider, R. J. (1993) Mol. Cell. Biol. 13, 1971-1980 [Abstract]
  55. Grafi, G., Sela, I., and Galili, G. (1993) Mol. Cell. Biol. 13, 3487-3493 [Abstract]
  56. Sullivan, N. F., and Willis, A. E. (1992) BioEssays 14, 333-336 [Medline] [Order article via Infotrieve]
  57. Seal, G., Brech, K., Karp, S. J., Cool, B. L., and Sirover, M. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2339-2343 [Abstract]
  58. Burd, C. G., Swanson, M. S., Görlach, M., and Dreyfuss, G. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9788-9792 [Abstract]
  59. Yang, S. T., and Deal, W. C., Jr. (1969) Biochemistry 8, 2806-2813 [Medline] [Order article via Infotrieve]
  60. Fukami-Kobayashi, K., Tomoda, S., and Go, M. (1993) FEBS Lett. 335, 289-293 [CrossRef][Medline] [Order article via Infotrieve]
  61. Mattaj, I. W. (1993) Cell 73, 837-840 [Medline] [Order article via Infotrieve]
  62. Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107 [Medline] [Order article via Infotrieve]
  63. Perona, J. J., Rould, M. A., Steitz, T. A., Risler, J., Zelwer, C., and Brunie, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2903-2907 [Abstract]
  64. Miseta, A., Woodley, C. L., Greenberg, J. R., and Slobin, L. I. (1991) J. Biol. Chem. 266, 19158-19161 [Abstract/Free Full Text]
  65. Furfine, C. S., and Velick, S. F. (1965) J. Biol. Chem. 240, 844-850 [Free Full Text]
  66. Mehlin, H., Daneholt, B., and Skoglund, U. (1992) Cell 69, 605-613 [Medline] [Order article via Infotrieve]
  67. Pinol-Roma, S., and Dreyfuss, G. (1992) Nature 335, 730-732
  68. Müller, W. E. G., Slor, H., Pfeifer, K., Hühn, P., Bek, A., Orsulic, S., Ushijama, H., and Schröder, H. C. (1992) J. Mol. Biol. 226, 721-733 [Medline] [Order article via Infotrieve]
  69. Pontius, B. W., and Berg, P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8403-8407 [Abstract]

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