Cloning, Sequencing, and Characterization of Alternatively Spliced Glutaredoxin 1 cDNA and Its Genomic Gene: CHROMOSOMAL LOCALIZATION, mRNA STABILITY, AND ORIGIN OF PSEUDOGENES

Jae B Park,Mark Levine

doi:10.1074/jbc.m412450200

Abstract

Alternatively spliced human glutaredoxin (Grx1(as)) cDNA was isolated from a neutrophil cDNA library, using a (32)P-labeled human glutaredoxin (Grx1) cDNA probe under non-stringent conditions. The sequence of Grx1(as) cDNA indicated that the open reading frame of the gene was identical to the open reading frame of the previously reported first human glutaredoxin (Grx1) cDNA, but the 3'-untranslated region of Grx1(as) was not homologous to Grx1 cDNA. Northern blot and RT-PCR analyses showed Grx1(as) mRNA was expressed in normal human neutrophils and transformed cells including U937, HL-60, THP, and Jurkat cells. Cloning and sequencing of the genomic gene corresponding to Grx1(as) cDNA showed that two different glutaredoxin cDNAs (Grx1(as) and Grx1) were generated from the same genomic gene via alternative splicing. Origination of Grx1(as) and Grx1 from the same gene was confirmed by chromosomal localization of the Grx1(as) gene to chromosome 5q13, the same location where the Grx1 gene was localized previously. During screening of the Grx1(as) genomic gene, two additional glutaredoxin pseudogenes were also isolated. Surprisingly, these pseudogenes contained 3'-untranslated regions that were nearly identical to the 3'-untranslated regions of Grx1(as,) not Grx1, cDNA. Because 3'-untranslated regions may be important in stabilizing mRNAs, the effect of the two 3'-untranslated regions of Grx1 and Grx1(as) on mRNA stability was investigated using luciferase reporter vectors with the 3'-untranslated regions. Luciferase activity was 2.6-fold greater in cells transfected with the reporter vector containing the 3'-untranslated region of Grx1(as) cDNA compared with the 3'-untranslated region of Grx1 cDNA. These data indicate that Grx1(as) cDNA is an alternatively spliced human Grx1 cDNA and that the Grx1(as) 3'-untranslated region may have a role in stabilizing mRNA.

Highlights

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
Isolation of Grx1as cDNA—To isolate different types of human glutaredoxin cDNA, partial cDNA glutaredoxin from nucleotides 50 to 270 of the open reading frame was used as a probe [3]
On comparing the sequences of mammalian glutaredoxin cDNAs, we found that swine glutaredoxin cDNA contains a 3Ј-untranslated region non-homologous to human Grx1 cDNA [18, 19]

Summary

EXPERIMENTAL PROCEDURES

Glutaredoxin (Grx, thioltransferase) is a small redox protein involved in oxidoreductive processes in cells through cat-. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF069668, NM002064, AF115104, AF115105, AF115106, HSU61726, and AY918930

The abbreviations used are

RESULTS

DISCUSSION