2. Ritsumeikan Global Innovation Research Organization (R-GIRO), Ritsumeikan University, Kusatsu, Shiga, Japan.
3. Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga, Japan.
4. Department of Surgery, Kansai Medical University, Hirakata, Osaka, Japan.
5. Present Address: World-Leading Drug Discovery Research Center, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Background: Natural antisense transcripts (asRNAs) are transcribed from many genes in various species. Recently, we found that asRNAs were transcribed from the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the essential inflammatory mediator nitric oxide. The iNOS asRNA corresponds to the 3'-untranslated region (3'UTR) of the iNOS mRNA. We also found that the iNOS asRNA stabilizes iNOS mRNA through mRNA-asRNA interactions. Single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence reduce iNOS mRNA levels by blocking mRNA-asRNA interactions; this knockdown method is referred to as natural antisense transcript-targeted regulation (NATRE). It is likely that asRNAs are transcribed from many other inducible genes.
Methods: To detect asRNAs complementary to the 3'UTR of inducible mRNAs, strand-specific reverse transcription-polymerase chain reaction (RT-PCR) was performed. When asRNA was expressed, sense oligonucleotides were designed on the basis of the secondary structure of the mRNA, and NATRE was applied to knockdown the asRNA.
Results: In rat hepatocytes treated with interleukin 1β (IL-1β), we found that asRNAs were transcribed from many inducible genes encoding inflammatory cytokines and chemokines, transcription factors, enzymes, and membrane proteins. When NATRE was applied to these genes, the sense oligonucleotides either down- or up-regulated the mRNA levels. These data support the hypothesis that asRNAs interact with mRNAs to regulate mRNA stability.
Conclusions: We found that asRNAs corresponding to mRNA 3'UTRs were transcribed from many IL-1β-inducible genes. Sense oligonucleotides using NATRE affected the mRNA levels of asRNA-expressing genes, suggesting that the asRNA-mediated regulation of mRNA stability plays a pivotal role. This mechanism may be a general post-transcriptional mechanism that is active during gene induction.
Keywords: antisense transcript, sense oligonucleotide, knockdown, cytokine, chemokine, NF-κB, TLR