Cloning, Sequencing, and Characterization of Alternatively SplicedGlutaredoxin 1 cDNA and Its Genomic GeneCHROMOSOMAL LOCALIZATION, mRNA STABILITY, AND ORIGIN OF PSEUDOGENES*

Received for publication, November 3, 2004, and in revised form, December 23, 2004Published, JBC Papers in Press, January 6, 2005, DOI 10.1074/jbc.M412450200

Jae B. Park‡ and Mark Levine§¶

From the ‡Phytonutrients Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service,United States Department of Agriculture, Beltsville, Maryland 20770 and the §Molecular and Clinical Nutrition Section,Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1372

Alternatively spliced human glutaredoxin (Grx1as)cDNA was isolated from a neutrophil cDNA library, us-ing a 32P-labeled human glutaredoxin (Grx1) cDNAprobe under non-stringent conditions. The sequence ofGrx1as cDNA indicated that the open reading frame ofthe gene was identical to the open reading frame of thepreviously reported first human glutaredoxin (Grx1)cDNA, but the 3�-untranslated region of Grx1as was nothomologous to Grx1 cDNA. Northern blot and RT-PCRanalyses showed Grx1as mRNA was expressed in normalhuman neutrophils and transformed cells includingU937, HL-60, THP, and Jurkat cells. Cloning and se-quencing of the genomic gene corresponding to Grx1ascDNA showed that two different glutaredoxin cDNAs(Grx1as and Grx1) were generated from the samegenomic gene via alternative splicing. Origination ofGrx1as and Grx1 from the same gene was confirmed bychromosomal localization of the Grx1as gene to chromo-some 5q13, the same location where the Grx1 gene waslocalized previously. During screening of the Grx1asgenomic gene, two additional glutaredoxin pseudogeneswere also isolated. Surprisingly, these pseudogenes con-tained 3�-untranslated regions that were nearly identi-cal to the 3�-untranslated regions of Grx1as, not Grx1,cDNA. Because 3�-untranslated regions may be impor-tant in stabilizing mRNAs, the effect of the two 3�-un-translated regions of Grx1 and Grx1as on mRNA stabilitywas investigated using luciferase reporter vectors withthe 3�-untranslated regions. Luciferase activity was 2.6-fold greater in cells transfected with the reporter vectorcontaining the 3�-untranslated region of Grx1as cDNAcompared with the 3�-untranslated region of Grx1 cDNA.These data indicate that Grx1as cDNA is an alternativelyspliced human Grx1 cDNA and that the Grx1as 3�-un-translated region may have a role in stabilizing mRNA.

Glutaredoxin (Grx,1 thioltransferase) is a small redox pro-tein involved in oxidoreductive processes in cells through cat-

alyzing disulfide-thiol exchange reactions (1–3). Thiol groups inproteins may act as redox-sensitive switches and are consid-ered to be a key element in maintaining cellular redox balance(4–8). Cellular redox imbalance induces radical oxygen speciesthat mediate signaling events leading to proliferation, apo-ptosis, and differentiation (4, 5). Because of its potential im-portance, the redox status of thiol groups is well balanced bybiological reducing molecules and proteins (6–10). Among re-dox proteins, glutaredoxin is a protein that regenerates S-thiolated cysteines in proteins that result from oxidative stress(11). Glutaredoxins have been isolated from prokaryotes andeukaryotes and have been proposed as redox proteins thatmediate several biological reactions (3, 11–16). Recently, asecond human glutaredoxin (glutaredoxin 2 or Grx2) has beencloned, and two alternatively spliced Grx2 mRNA isoformswere identified (17).

In our laboratory, we searched for mRNA isoforms of the firsthuman glutaredoxin (glutaredoxin 1 or Grx1), because swineglutaredoxin cDNA contains a 3�-untranslated region that hasno counterpart in human glutaredoxin 1 cDNAs (18, 19). Ahuman neutrophil library was screened with 32P-labeled Grx1cDNA using non-stringent conditions. Screening yielded a newhuman glutaredoxin cDNA, Grx1as. Its nucleotide sequencewas identical to the open reading frame of Grx1 cDNA, but the3�-untranslated region sequence was comparable to that ofswine glutaredoxin cDNA rather than that of human Grx1cDNA. Northern blots and RT-PCR were performed to verifyGrx1as mRNA in several human cells. Genomic cloning andchromosomal localization of Grx1as were performed to deter-mine genomic origins of Grx1 and Grx1as cDNAs. Finally ef-fects of 3�- untranslated regions from Grx1as and Grx1 cDNAson mRNA stability were investigated.

EXPERIMENTAL PROCEDURES

Materials—Cloned human Grx1 cDNA (3) was maintained in Esch-erichia coli strain JM101. Restriction endonuclease, T4 DNA ligase, T4polynucleotide kinase, and avian myeloblastosis virus reverse tran-scriptase were purchased from Promega (San Luis Obispo, CA). Taqpolymerase and deoxynucleotides were obtained from PerkinElmer LifeSciences and radiolabeled nucleotides from Amersham Biosciences. Ahuman monocyte cDNA library was purchased from Clontech (catalogno. HL1056B, Palo Alto, CA). Ascorbic acid, EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Tris-HCl, and dithiothreitol were pur-chased from Sigma.

Construction of Human Neutrophil cDNA and Genomic Library—Ahuman neutrophil cDNA library was constructed as described previ-ously (3). Briefly, first and second strand cDNA were synthesized(Stratagene, La Jolla, CA) using 5 �g of human neutrophil mRNAobtained from 10 subjects, each of whom provided �1 � 109 neutrophilsisolated by apheresis (20, 21). The cDNAs were ligated to Uni-ZAP

* This work was supported in part by Grant Z01 DK 54506 fromNIDDK, National Institutes of Health. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF069668,NM002064, AF115104, AF115105, AF115106, HSU61726, andAY918930.

¶ To whom correspondence should be addressed: Molecular and Clin-ical Nutrition Section, Bldg. 10, Rm. 4D52 MSC-1372, National Insti-tutes of Health, Bethesda, MD 20892-1372. Tel.: 301-402-5588; Fax:301-402-6436; E-mail: markL@mail.nih.gov.

1 The abbreviations used are: Grx, glutaredoxin; Grx1, human glu-taredoxin cDNA; Grx1as, alternatively spliced human glutaredoxin

cDNA; RT-PCR, reverse transcriptase polymerase chain reaction;MV, cytomegalovirus.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 11, Issue of March 18, pp. 10427–10434, 2005Printed in U.S.A.

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vector and transfected into XL-Blue strain using Gigapack (Strat-agene). Human neutrophil genomic library was constructed as de-scribed previously (18). Neutrophil genomic DNA was isolated per man-ufacturer’s instructions (Qiagen, Valencia, CA). Isolated DNA waspartially digested with Sau3A, and size 6–15 kb DNA was isolatedusing sucrose gradient centrifugation. The isolated DNA was ligated to� DASHII arms predigested with BamHI (Stratagene). The ligated �was in vitro packaged using Gigapack II Gold (Stratagene), plated,and amplified.

Isolation and Sequencing of Alternatively Spliced Human Glutare-doxin (Grx1as) cDNA—An amplified human neutrophil cDNA librarywas screened with random-primed 32P-labeled partial human neutro-phil glutaredoxin cDNA from nucleotides 50 to 270 of the open readingframe (3). Several clones were selected from 2 � 106 recombinants, andclones were further purified by sequential platings. The size and integ-rity of cDNA was determined by PCR with primers corresponding tovarious regions of the human cDNA. Nucleotide sequences were deter-mined using the dideoxy chain termination method with modified T7DNA polymerase (Amersham Biosciences).

Northern Blot of Grx1as cDNA—Northern blot was performed using 2�g of poly(A)� RNAs isolated from neutrophils, U937, HL-60, PLB, andJurkat cells. The blot was hybridized under stringent conditions (22)with a probe of the 3�-untranslated region of Grx1as cDNA.

Isolation and Sequencing of Genomic Grx1as Gene—An amplifiedhuman neutrophil genomic DNA library was screened with random-primed 32P-labeled Grx1as 3�-untranslated region cDNA. Two overlap-ping positive clones were selected from 8 � 106 recombinants. Theplaques were purified and designated G1-G2. The size and integrity ofinsert genomic DNA was determined by PCR with primers correspond-ing to various regions of the human cDNA. PCR reactions were per-formed with a DNA Thermal Cycler according to the manufacturer’srecommendations (PerkinElmer Life Sciences). One (G1) of two clonescontained the entire glutaredoxin gene, the 1.2-kb upstream 5�-flankingregion and the 3-kb downstream 3�-flanking region. A genomic frag-ment of �10 kb in clone G1 was analyzed with PCR. The DNA contain-ing the glutaredoxin promoter region was amplified using the 18 oli-gomer (5�-CACAAACTCTTGAGCCAT; primer 1) of the antisensestrand complementary to nucleotide positions 1–18 of glutaredoxincDNA, and T3 primer located in the left � arm. Glutaredoxin genomicgene was digested with SacI, and the sequences of digested DNA frag-ments were determined using the dideoxy chain termination method

with modified T7 DNA polymerase and the ALF DNA sequencer (Am-ersham Biosciences). During the genomic screening, we also isolatedtwo glutaredoxin pseudogenes (GS1 and GS2).

Chromosomal Localization—The Grx1as gene was labeled withdigoxigenin dUTP by nick translation. Labeled probe was combinedwith sheared human DNA and hybridized to normal metaphase chro-mosomes derived from phytohemagglutinin-stimulated peripheralblood lymphocytes in a solution containing 50% formamide, 10% dex-tran sulfate, and 2� sodium chloride, sodium citrate buffer (23).

Determination of Effect of 3�-Untranslated Regions on mRNA Stabil-ity—To measure the effect of short and long 3�- untranslated regions onmRNA stability, a luciferase reporter vector (pCMV-Luc) was con-structed by cloning PCR-amplified luciferase gene into pCR 3.1 (Invitro-gen). The reporter vectors contained either the short or long 3�-untrans-lated region of human glutaredoxin: pCMV-Luc-3�-untranslated regionGrx1 or pCMV-Luc-3�-untranslated region Grx1as, respectively. Thevectors were constructed by insertion of each 3�-untranslated regiondownstream of the luciferase gene using the PstI site. Vectors weretransfected with calcium phosphate into HeLa cells. Cells were culturedin Dulbecco’s modified Eagle’s medium supplemented with 10% fetalcalf serum. Cells were grown to �50% confluence in 60-mm Petridishes. The two reporter plasmids were individually transfected intocells using calcium phosphate. After transfection, cells were incubatedfor an additional 48 h, harvested, and lysed. Luciferase assays of thelysates were performed according to the manufacturer’s protocol (Pro-mega). Transfection efficiency was monitored by co-transfection withpSEAP-2 promoter vector, the alkaline phosphatase activity of whichwas determined according to the manufacturer’s protocol (Clontech).

Isolation of Grx1as and Grx1 Recombinant Proteins—Grx1 was am-plified and purified as described (3). Grx1as cDNA was amplified byPCR using two primers: 5�-ATGGCTCAAGAGTTTGTG-3� and 5�-TTACTGCAGAGCTCCAAT-3�, which were complementary to nucleo-tide positions 1–18 (sense) and 303–321 (antisense) of glutaredoxincDNA, respectively. For expression, PCR fragments were cloned intopGEX expression vector (Amersham Biosciences). The vector was trans-formed into E. coli (BL21), and bacteria were cultured overnight at37 °C in Luria Bertani medium containing ampicillin (100 �g/ml). Over-night culture was inoculated into fresh medium and cultured furtherwith vigorous shaking. When OD600 nm was 0.6, protein expression wasinduced by adding 0.5 mM isopropyl �-D-thiogalactopyranoside. After4 h of induction, bacteria were collected by centrifugation. Recombinant

FIG. 1. The nucleotide sequenceand deduced amino acid sequence ofGrx1as glutaredoxin cDNA. Sequences1 and 2 represent Grx1as and Grx1, re-spectively. The nucleotides are numbered5� to 3�, and amino acids are numberedfrom N to C termini, respectively. Twoconserved regions containing a total offour cysteines are underlined. Bold nucle-otides indicates that they are the same inboth clones, and *** indicates the stopcodon.

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glutathione was expressed as glutathione S-transferase fusion protein,and the fusion protein was purified using a glutathione-Sepharosecolumn (Amersham Biosciences).

Dehydroascorbic Acid Reducing Activity—Reducing activity was de-termined in 25 �l of 100 mM Tris-HCl buffer (pH 7.5) containing (finalconcentrations) 0.8 mM reduced glutathione, 500 �M of dehydroascorbicacid, and appropriate amounts of enzyme. The reaction was initiated byadding dehydroascorbic acid and measured for 3 min at room temper-ature. The reaction was terminated by addition of 35 �l of 90% meth-anol in water containing 1 mM EDTA. The final mixture was centri-fuged for 10 min at 14,000 � g, and the supernatant immediatelyanalyzed by high performance liquid chromatography (24). A chemicalreaction containing no enzyme was simultaneously measured under thesame conditions. Where indicated chemical activity was subtractedfrom total reducing activity to yield enzymatic activity. Chemical activ-ity was always measured and accounted for (3). The amount of reduceddehydroascorbic acid was determined as ascorbic acid by high perform-ance liquid chromatography with coulometric electrochemical detectionas described previously (24). Dehydroascorbic acid was always preparedfresh immediately prior to experiments as described (25).

Western Blot—Western blot analysis was performed using anti-glu-taredoxin serum, as described (3, 26). Protein samples were electro-phoresed in 10–20% gradient SDS gels, then electroblotted onto anitrocellulose membrane.

RESULTS

Isolation of Grx1as cDNA—To isolate different types of hu-man glutaredoxin cDNA, partial cDNA glutaredoxin from nu-cleotides 50 to 270 of the open reading frame was used as aprobe (3). This region was selected because it contained fourconserved cysteines including an active site cysteine. Plaques(8 � 106) were screened under low stringent conditions withthe 32P-labeled DNA probe. Positive plaques were selected, andthe integrity of each plaque was tested by PCR, using internal

primers located in the previously reported glutaredoxin cDNA(3, 27, 28) (data not shown). The nucleotide sequence of theisolated clone was determined and compared with that of Grx1cDNA (Fig. 1). Surprisingly, the nucleotide sequences of thetranslation regions of both cDNAs were homologous, but the3�-untranslated region of the newly isolated cDNA was differ-ent from that of Grx1 cDNA. To date, at least three mammalianglutaredoxin cDNAs have been reported, among which is swineglutaredoxin cDNA. Swine glutaredoxin cDNA contains atranslation region exhibiting �80% identity to the humancounterpart, but a region with no similarity is found in the3�-untranslated regions of human and swine glutaredoxincDNAs (19). The newly isolated glutaredoxin cDNA not onlyexhibited an identical nucleotide sequence to that of previouslyreported human glutaredoxin (Grx1) cDNA in the translationregion, but also was quite similar to the 3�-untranslated regioncounterpart of swine glutaredoxin cDNA rather than Grx1cDNA. We termed the newly isolated glutaredoxin cDNAGrx1as.

Grx1as Is Present in Multiple Cell Types—Expression ofGrx1as mRNA was determined by Northern blot and RT-PCRwith mRNAs from human neutrophils, U937, HL-60, THP, andJurkat cells. Transfers of mRNAs onto nitrocellulose mem-brane were performed using a standard method (22). The probeused was not a full sequence of Grx1as cDNA, because thetranslation region of Grx1as cDNA contained the same nucleo-tide sequence as Grx1 cDNA. The probe was amplified by PCRwith the two primers (forward and reverse primers located atnucleotides 350–370 and 520–540, respectively in Fig. 1). The

FIG. 2. Northern blot of Grx1as glutaredoxin cDNA. 10 �g oftotal RNA from neutrophils (lane 1), HL-60 cells (lane 2), THP cells(lane 3), PLB cells (lane 4), and Jurkat cells (lane 5) were transferredonto nitrocellulose membrane, and hybridized using the probe of the3�-untranslated region of Grx1as. Glyceraldehyde-3-phosphate dehydro-genase controls are shown at the bottom of the figure.

FIG. 3. Western blots of recombinant proteins expressed fromGrx1 and Grx1as. Recombinant proteins were made and purified asdescribed under “Experimental Procedures,” and polyclonal antibodywas used for detection. The recombinant protein (glutaredoxin) fromGrx1 is shown in lane A, and the recombinant protein from Grx1as isshown in lane B.

TABLE IBiochemical and kinetic analyses of dehydroascorbic acid reducing

activities of expressed Grx1 and Grx1as

Activities were measured as described under “Experimental Proce-dures.”

RecombinantGrx1

RecombinantGrx1as

Optimal pH 7.5 7.5Km for glutathione (mM) 2.2 2.0Km for dehydroascorbic acid (�M) 220 240Specific activity (umol/min/mg) 3 3

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amplified DNA fragment that had no homology to Grx1 cDNAwas 32P-labeled by the random primer method. The transferrednitrocellulose membrane was hybridized using stringent con-ditions with the 32P-labeled probe. Grx1as cDNA was expressedin all the cells tested (Fig. 2). The data indicate that Grx1as isexpressed in cells of myelocytic and lymphocytic origins.

There are several indications that Grx1as is a generally ob-served phenomenon and not an artifact from a single subjectsource. The library from which Grx1as was originally isolatedwas prepared from neutrophils from 10 subjects. As shown inFig. 2, Grx1as expression was verified by Northern blot in allcells tested of myeloid origin: HL60 cells, THP cells, Jurkatcells, and PLB cells. As confirmation of these data, RT-PCR

with specific primers for Grx1as showed that the mRNA waspresent in all the myeloid cell types tested (data not shown).Finally, when Grx1as specific primers were used, the mRNAwas also present in a commercial myeloid library.

Characterization of Recombinant Grx1as—The nucleotide se-quences of Grx1as and Grx1 cDNAs are identical in their trans-lation regions. Therefore, we predicted that the recombinantprotein from Grx1as cDNA would be likely to exhibit the samebiochemical and kinetics properties as glutaredoxin from Grx1cDNA. To study this, the translation region (open readingframe) of Grx1as cDNA was amplified and ligated into pGEXfusion expression vector as described under “Experimental Pro-cedures.” Glutathione S-transferase-fused protein was purified

FIG. 4. The nucleotide sequence of asubclone of Grx1as genomic gene. Thedonor sites of intron junctions are markedwith slash marks. The sequence of the3�-untranslated region of Grx1as cDNA(as shown in Fig. 1) is underlined, and thesequence of the 3�-untranslated region ofGrx1 originally reported (14) is in boldtype.

FIG. 5. The nucleotide sequence ofthe genomic gene of Grx1as cDNA.The donor sites of the intron junctions aremarked with slash marks. The sequenceof 3�-untranslated region of Grx1as cDNAis underlined. In the upper half of thefigure, the glutaredoxin open readingframe is in bold type and the two regionscontaining a total of four cysteines areunderlined. In the lower half of the figure,bold type indicates identity between Grx1and Grx1as 3�-untranslated regions, anddouble underlining indicates the signalsequence for the poly(A) tail.

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using a glutathione-Sepharose column, and the fusion proteinwas digested with thrombin (0.1%, w/w) to yield recombinantprotein. Glutaredoxin, from Grx1 cDNA, was isolated and pu-rified as described (3).

Biochemical properties of the recombinant proteins fromGrx1 and Grx1as cDNAs were characterized with respect todehydroascorbic acid reducing activities (Table I). The follow-ing properties were virtually identical: apparent Km for gluta-thione; apparent Km for dehydroascorbic acid; optimal pH; andspecific activity. Each recombinant purified protein was de-tected by Western blot (Fig. 3), using polyclonal antibody pre-pared by injecting Grx1 in rabbits (3). Taken together, thesedata indicate that biochemical and kinetics properties of thetwo proteins were indistinguishable.

Isolation and Sequencing of the Genomic Gene of Grx1as—Todelineate the origin of Grx1as cDNA, its genomic gene wasscreened in a neutrophil genomic library with the same probeused in the Northern blot. A positive plaque was selected as thecandidate for the genomic gene of Grx1as cDNA. The integrityand authenticity of the clone were checked by PCR using sev-eral primers (see “Experimental Procedures”). To obtain de-tailed information on the nucleotide sequence of the isolatedgenomic gene, the clone was digested with restriction enzymesand subcloned into pGEM sequence vector. Surprisingly, thenucleotide sequence of the 5�-flanking region of Grx1as cDNAwas exactly the same as that of the published genomic gene ofGrx1 cDNA (17, 18). Due to this unexpected result, the nucleo-tides of other regions of the isolated genomic gene were furthersequenced. We found that the sequenced regions of the Grx1as

genomic gene were perfectly matched to the correspondingregion of the Grx1 genomic gene (17, 18). Therefore, everyindividual subclone was amplified by PCR with specific prim-ers used in the Northern blot in order to identify the subclonecontaining the unique 3�-untranslated region of Grx1as cDNA.One subclone was confirmed by PCR to contain the 3�-untrans-lated region, and sequenced for its verification. For clarity, apartial nucleotide sequence of this subclone is shown in Fig. 4.The subclone contained, in order as shown: an overlappingpartial sequence of an intron; the 3�-untranslated region ofGrx1as cDNA (as shown in Fig. 1); a unique 3�-untranslatedregion of Grx1as cDNA; and the 3�-untranslated region of Grx1cDNA (also as shown in Fig. 1). The full sequence of thissubclone is shown in Fig. 5 and represents the nucleotidesequence of the genomic gene of Grx1as. The sequence includesthe coding region that is identical to glutaredoxin, and containstwo introns and three exons. This gene organization is identicalto that of the genomic gene for Grx1 (17, 18) (Fig. 6). Takentogether, these data indicate that Grx1 and Grx1as cDNAs areproduced from the same human glutaredoxin genomic gene viaalternative splicing at gggcag/AACAGGCCC in the second in-

tron instead of ccacag/ATCTCATAG (Figs. 5 and 6). Thus,Grx1as cDNA, but not Grx1, includes an additional 566 nucleo-tides from second intron in its 3�-untranslated region. Thealternative splicing of human glutaredoxin genomic gene ex-plains why human Grx1as and swine glutaredoxin cDNAs, butnot Grx1 cDNA, are similar in their 3�-untranslated regions. Toconfirm this, a full-length Grx1as cDNA was cloned and com-pletely sequenced. Sequencing the human glutaredoxingenomic gene showed that an isolated full-length cDNA ofGrx1as contained the unique sequence of 3�-untranslated re-gion of Grx1as, and 3�-untranslated region of Grx1 (Fig. 7). The3�-untranslated region of Grx1as cDNA is approximately twicethe size of the counterpart of the 3�-untranslated region ofGrx1 cDNA.

Chromosomal Localization of the Grx1as Gene—The se-quence of the genes from Grx1 and Grx1as cDNAs indicatedthat each individual human glutaredoxin cDNA was probablytranscribed from the same gene. This was confirmed by chro-mosomal localization of the isolated Grx1as gene. Specific hy-bridization signals were detected by incubating the hybridizedslides in fluoresceinated antidioxigenin antibodies followed bycounterstaining with 4�-6-diamidino-2-phenylindole dihydro-chloride. The results showed a specific labeling of chromosome5, based on size, morphology, and banding pattern. In addi-tional experiments, a genomic probe (previously mapped to5q32 and confirmed by co-hybridization with a probe from thecri du chat locus on chromosome arm 5p) was co-hybridizedwith the hybridized probe of the Grx1as gene. In these experi-ments the middle and distal long arm of chromosome 5 werespecifically labeled. Measurement of ten specifically hybridizedchromosomes 5 demonstrated that the glutaredoxin gene islocated at a position which is 39% of the distance from thecentromere to the telomere of chromosome arm 5q, an areawhich corresponds to the boundary between bands 5q15 and5q21 (Fig. 8). This chromosomal position was previouslymapped to the human Grx1 gene (29). The nucleotide sequenceand chromosomal mapping of the human Grx1as gene indicatethat Grx1as cDNA is undoubtedly transcribed from the samegene that human Grx1 cDNA comes from.

Effect of Two 3�-Untranslated regions of Grx1 and Grx1as onTheir mRNA Stability—During the screening of the Grx1genomic gene, two different putative pseudogenes (GS1 andGS2) were isolated and sequenced. They seemed to displaytypical characteristics of pseudogenes: no intron, poly(A) tail-ing, and sporadic mutations. However, some regions of the3�-untranslated regions of these genes were not homologous tothe 3�-untranslated region of Grx1 cDNA. Instead, Grx1as

cDNA and the two glutaredoxin pseudogenes were very similarin portions of their 3�-untranslated regions (Fig. 9). It wassurprising that the pseudogenes had greater resemblance to

FIG. 6. Physical map and compari-son of an isolated gene of Grx1as withGrx1 gene (17). Grx1 and Grx1as are de-picted in I and II, respectively. The se-quences of the junction regions betweenexons and introns are shown in III. TwocDNAs of Grx1 (I) and Grx1as (II) are com-pared side by side to illustrate thatthe 3�-untranslated regions are not iden-tical to each other. Different junctions be-tween exons and introns are labeledalphabetically.

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Grx1as than to Grx1 cDNA, because pseudogenes are commonlyconsidered to originate from their authentic mRNA. Stability ofmRNA may therefore be a contributing factor for generatingpseudogenes. We investigated this possibility by measuring theeffect of the 3�-untranslated regions of Grx1 and Grx1as on thestability of their mRNAs. The effect of the two 3�-untranslatedregions was determined by constructing the vectors pCMV-Luc-3�-untranslated region Grx1 and pCMV-Luc-3�-untrans-lated region Grx1as, transfecting these vectors into cells, andmeasuring luciferase activity (see “Experimental Procedures”).As shown in Table II, the decreasing order of luciferase activ-ities was pCMV-Luc-3�-untranslated region Grx1as � pCMV-

Luc-3�-untranslated region Grx1 � pCMV-Luc control. Sinceall three reporter vectors have the same CMV promoter, thepromoter activities of the vectors and their initial mRNAs maybe very similar. To verify this assumption, quantitative RT-PCR was performed with mRNAs from HeLa cells transfectedwith each reporter vector. Amounts of luciferase mRNA fromthe three transfected cells was very similar (data not shown).These data indicate that the quantity of luciferase mRNAs wassimilar in the cells transfected with three different reportervectors, but mRNA containing 3�-untranslated region Grx1as

might be more stable than the other two mRNAs, therebyproducing more luciferase. Because the 3�-untranslated regionof Grx1as mRNA may confer enhanced stability, Grx1as mRNAmay be a better template than Grx1 mRNA for generatingits pseudogenes.

DISCUSSION

On comparing the sequences of mammalian glutaredoxincDNAs, we found that swine glutaredoxin cDNA contains a3�-untranslated region non-homologous to human Grx1 cDNA(18, 19). The discrepancy between human and swine cDNAssuggested that human glutaredoxin cDNA might exist in morethan one form. It seemed reasonable that the existence ofdifferent human glutaredoxin cDNAs and a non-homologousregion in swine glutaredoxin cDNA should be addressed priorto investigating glutaredoxin regulation, because it is possiblethat each glutaredoxin cDNA may be regulated differently inhuman cells. As a way to search for different human glutare-doxin mRNA, a human neutrophil cDNA library was screenedwith a probe of Grx1 cDNA using non-stringent conditions. Inthe screening process, a different human glutaredoxin (Grx1as)cDNA was isolated. Surprisingly, the nucleotide sequence andchromosomal localization of the genomic gene of Grx1as cDNAindicated that Grx1as and Grx1 were transcriptional productsderived from the same gene via alternative splicing. The isola-tion, sequencing, and localization of Grx1as cDNA provide ananswer for the lack of identity between the 3�-untranslatedregion of human glutaredoxin (Grx1) and swine glutaredoxin.

However, it was still not clear whether two different glutare-doxin cDNAs resulted from a single transcript with alternative

FIG. 7. Comparison of nucleotide sequences of full-lengthcDNAs of Grx1as and Grx1. Nucleotides are numbered 5� to 3�, andamino acids are numbered from the N to C termini, respectively. Twoconserved regions containing a total of four cysteines are underlined.The homologous regions are in bold, and the signal sequence for thepoly(A) tail is double underlined.

FIG. 8. Chromosomal localization of Grx1as gene by fluores-cence in situ hybridization (FISH). Normal metaphase chromo-somes from peripheral blood lymphocytes were hybridized with antid-ioxigenin-labeled probe. A specific hybridization signal was detected,indicated as the top mark (G), and a cohybridization signal, indicatedwith the bottom mark (C) in the figure. The signal position was locatedin 5q15, demonstrated in the ideogram of chromosome 5.

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splicing or from two different transcripts generated using twodifferent promoter regions of the same gene. Primer extensionand S1 mapping experiments answered this question (18). Hu-man neutrophil mRNA exhibited only one band protected fromS1 nuclease, even though neutrophils contain two differentglutaredoxin mRNAs (18) (Fig. 2). These data suggest that onepromoter is apparently used for expression of a pre-mRNAleading to two different human glutaredoxin mRNAs via alter-native splicing. Determining how this process proceeds mayadvance understanding of the cellular response to oxidativestress. In our preliminary studies, a certain region of the pro-moter of human glutaredoxin gene was identified as a tran-scriptional regulatory region, which may bind to several poten-tial transcriptional factors. Future investigation will providedetailed information regarding the regulation of human glu-taredoxin via this transcriptional regulatory region.

When we began this study, it was unclear whether isoformsof human glutaredoxin existed. However, human glutaredoxin2 (Grx2) has been isolated recently, and two mRNAs of Grx2have been identified as alternative splicing products (17). In-

terestingly, both Grx1 and Grx2 have two mRNAs generatedvia alternative splicing, respectively. During our screening ofthe human glutaredoxin 1 gene, two additional genes wereisolated and sequenced. Each of these genes has characteristicsof a pseudogene: non-existence of introns, possession of poly(A)tail, and sporadic mutations (30). Of interest, the isolated pseu-dogenes (GS1 and GS2) resemble Grx1as cDNA rather thanGrx1 cDNA. In other words, each pseudogene contains a 3�-untranslated region similar to the 3�-untranslated region ofhuman Grx1as cDNA. Furthermore, a glutaredoxin pseudogene(GS1) has a mutation of the first conserved cysteine to pheny-lalanine. Further investigation of this pseudogene is now un-derway to determine whether the pseudogene is expressed incells, and what biological function the pseudogene may deliver,if expressed. Although alternative splicing is a common mech-anism used to generate two mRNAs in Grx1 and Grx2, respec-tively, the biological consequences of the 3�-untranslated re-gions of Grx2 are largely unknown currently.

In this report, two different human glutaredoxin 1 mRNAswere demonstrated to exist, and potential biological conse-quences of the two mRNAs were elucidated. Glutaredoxin pseu-dogenes contain a long 3�-untranslated region, similar to thatof Grx1as cDNA. According to current hypotheses, mRNAs aretemplates for producing pseudogenes, and pseudogenes containa poly(A) tail, sporadic mutations, and no introns. If so, humanGrx1as cDNA with a long 3�-untranslated region was perhapsmore stable than Grx1 cDNA with a short 3�-untranslatedregion. Greater stability would increase the likelihood ofGrx1as serving as a template for human glutaredoxin pseudo-genes. This study showed that a luciferase gene with the longer3�-untranslated region of Grx1as had a more stable transcript

FIG. 9. Nucleotide sequence comparison of Grx1as cDNA with two glutaredoxin pseudogenes (GS1 and GS2). The deduced amino acidsequence of Grx1as is depicted on the top line. Sequences 1, 2, and 3 represent glutaredoxin Ias, glutaredoxin pseudogene I (GS1), and glutaredoxinpseudogene II (GS2), respectively. The 3�-untranslated region unique to Grx1as is in bold. Shaded areas in the pseudogenes represent nucleotideidentity to the 3�-untranslated region unique to Grx1as. Two cysteine regions are underlined; the region indicating stop codon is indicated by ***,and the signal sequence for the poly(A) tail is double underlined.

TABLE IIEffect of 3�-untranslated regions of Grx1 and Grx1ast on mRNA

stability as measured by luciferase activityEach 3�-untranslated region was fused to luciferase as described

under “Experimental Procedures,” and pCMV-Luc was the control re-porter vector. Each of the three vectors was transfected into HeLa cells,and luciferase activities were determined as described under “Experi-mental Procedures.”

PCMV-Luccontrol

pCMV-Luc-3�-untranslated

region Grx1

pCMV-Luc-3�-untranslatedregion Grx1as

Luciferase activity 150 � 70 648 � 150 1711 � 180

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than the shorter 3�-untranslated region of Grx1, as indicatedby greater luciferase activity in transfected cells. In summary,the data in this paper provided information about the expres-sion of different human glutaredoxin 1 cDNAs and their mRNAstability, and also may provide insight about the origin andpotential biological consequence of the 3�-untranslated regionfrom Grx2.

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Jae B. Park and Mark LevineSTABILITY, AND ORIGIN OF PSEUDOGENES

cDNA and Its Genomic Gene: CHROMOSOMAL LOCALIZATION, mRNA Cloning, Sequencing, and Characterization of Alternatively Spliced Glutaredoxin 1

doi: 10.1074/jbc.M412450200 originally published online January 6, 20052005, 280:10427-10434.J. Biol. Chem. 

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