Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes.
During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1beta (IL-1beta) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3'-untranslated region (3'UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5-kilobase region 3'-flanking to iNOS gene exon 27 was involved in IL-1beta induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3'UTR. These results together with the data in a yeast RNA-hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU-rich element-binding protein HuR, a human homolog of embryonic lethal-abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA-binding proteins may mutually interact.


CONCLUSIONS
The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA-RNA interaction may be a new therapeutic target for NO-mediating inflammatory diseases.
Matsui
Kosuke
K
Department of Surgery, Kansai Medical University, Moriguchi, Osaka, Japan.
Nishizawa
Mikio
M
Ozaki
Takashi
T
Kimura
Tominori
T
Hashimoto
Iwao
I
Yamada
Masanori
M
Kaibori
Masaki
M
Kamiyama
Yasuo
Y
Ito
Seiji
S
Okumura
Tadayoshi
T
D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers; D017930:Genes, Reporter; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007249:Inflammation / Q000201:enzymology / Q000235:genetics; D053583:Interleukin-1beta / Q000494:pharmacology; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D017931:DNA Primers; D053583:Interleukin-1beta; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D009569:Nitric Oxide; D012313:RNA; D052247:Nitric Oxide Synthase Type II