Identification and expression of deoxyribonuclease (DNase) I alternative transcripts in the rat.
Deoxyribonuclease (DNase) I has been implicated in the induction of DNA fragmentation and cell death, however little is known about its regulation in vivo. In the present study, we describe that DNase I messenger RNA (mRNA) is alternatively spliced in rat kidney, and the activity of the DNase I correlates with the alternative splicing during the course of renal ischemia/reperfusion. Northern blot analysis with mRNA from control rat kidneys and kidneys subjected to ischemia/reperfusion in vivo yielded two bands of approximately 1.3 and 1.5 kb, suggesting the possibility of alternative splicing. However, prolonged reperfusion up to 16 h resulted in the predominant expression of 1.3 kb transcript. The disappearance of the 1.5 kDa band was associated with the increased DNase I activity in the kidney during ischemia/reperfusion. To study the alternative splicing of the DNase I mRNA, rat kidney cortex DNA complementary to RNA library was screened using rat DNase I probe. Twenty-one positive clones were obtained and were compared with the reported DNase I mRNA transcript cloned from rat parotid gland. All clones showed 100% homology with the reported DNase I coding sequence and part of 5'-untranslated region (5'-UTR), named exon 1a by us. Twelve out of 21 isolated clones had longer 5'-UTR than previously described, and DNase I pre-mRNA was alternatively spliced in this region. Six out of these 12 clones contained extra up to 153 bp in extreme 5' end, whereas, in six other clones, an internal 132 bp segment (exon 1b) of this additional sequence was absent, and only the extreme 5'UTR sequence (exon 1c) was found in these clones. The nucleotide analysis showed that alternating exon 1b has the possibility of a secondary structure with high internal homology and potential for at least one major stable stem-loop. Both newly identified segments, exons 1b and 1c, were also identified in genomic DNA. The long splice variant, containing exon 1b, is expressed only in the kidney among different tissues tested. Exon 1b inhibited translational activity of DNase I mRNA in vitro. Our data suggest that alternative splicing in 5'-UTR in the kidney may provide a prompt DNA-independent regulation of DNase I activity when DNA is damaged during ischemic injury.
Basnakian
Alexei G
AG
Department of Internal Medicine, Division of Nephrology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 501, Little Rock, AR 72205, USA. basnakianalexeig@uams.edu
Singh
Amar B
AB
Shah
Sudhir V
SV
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000235:genetics*; D005091:Exons / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D007668:Kidney / Q000098:blood supply / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000503:physiopathology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger; D004247:DNA; D003850:Deoxyribonuclease I