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Title: CLONING, SEQUENCING, AND CHARACTERIZATION OF ALTERNATIVELY SPLICED GLUTAREDOXIN CDNA AND ITS GENOMIC GENE

Author
item Park, Jae
item LEVIN, MARK - NIH, BETHESDA, MD

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2004
Publication Date: 2/1/2005
Citation: Park, J.B., Levin, M. 2005. Cloning, sequencing, and characterization of alternatively spliced glutaredoxin cDNA and its genomic gene. Journal of Biological Chemistry. 280:10427-10434.

Interpretive Summary: Glutaredoxin (thioltransferase) is a small redox protein that plays an important role in the redox balance in cells. Cellular redox balance is considered to be a key mechanism in maintaining homeostasis of cells. Mixed disulfide formation in proteins is significantly increased in cells under oxidative stress. Glutaredoxin is the most efficient protein in regenerating S-thiolated cysteines in proteins resulting from oxidative stress. Glutaredoxin is also involved in vitamin C recycling and other cellular events. Recently, glutaredoxin is even reported to regulate the activity of glutathionylated HIV-1 protease. In this report, a new human glutaredoxin (GRXas) cDNA and its gene were cloned and sequenced, and the expression of GRXas mRNA was confirmed in neutrophils and other cells. This study reveals that cDNAs of GRX and GRXas originate from one genomic gene via alternative splicing, and 3'-UTR of GRXas mRNA is involved in enhancing the stability of its mRNA. Oxidative stress is a common cause of many chronic diseases such as inflammation, heart disease, and cancer. Glutaredoxin is an important redox protein protecting cysteine residues in proteins from harmful oxidant attacks. The outcomes of current and future studies will provide researchers in nutrition and molecular biology fields with information regarding genomic regulation of the new glutaredxoin expression, and its cellular and molecular actions related to human diseases.

Technical Abstract: Alternatively spliced human glutaredoxin (GRXas) cDNA was isolated from a neutrophil cDNA library using the 32P-labeled glutaredoxin cDNA probe in non-stringent conditions. The sequence of this GRXas cDNA indicates that the open reading frame (ORF) of the gene is identical to the ORF of the previously reported human glutaredoxin (GRX) cDNA, but the 3'-untranslated region (UTR) of GRXas is non-homologous to GRX cDNA. This unique 3'-UTR of GRXas cDNA resembles the counterpart of swine glutaredoxin cDNA. The expression of GRXas mRNA was demonstrated by Northern Blot and RT-PCR. The cloning and sequencing of a genomic gene corresponding to GRXas cDNA showed that two different glutaredoxin cDNAs (GRXas and GRX) were generated from the same genomic gene via alternative splicing, thereby explaining why the 3'-UTR of human GRX is different from that of human GRXas. This fact was confirmed by chromosomal localization of the GRXas gene, mapped to chromosome 5q13, at which the GRX gene was previously localized. During the screening of the GRXas genomic gene, two glutaredoxin pseudogenes were isolated. Surprisingly, these pseudogenes contained 3'-UTR exhibiting high homology to 3'-UTR of GRXas, not GRX, cDNA. Because pseudogenes originate from their authentic mRNAs, the effect of the two 3'-UTRs of GRX and GRXas on mRNA stability was investigated using luciferase reporter vectors with the 3'-UTRs. The activity of luciferase more than doubled in the cells transfected with the reporter vector containing the 3'-UTR of GRXas cDNA compared with the transfected cells with the vector with 3'-UTR of GRX cDNA. The data indicates 3'-UTR of GRXas might be important in conferring stability to its mRNA.