Transcriptional Regulation Of A Novel Splicing Isoform Int2v1 Of Interferon Regulatory Factor 3

Interferon regulatory factor 3 (IRF-3) is one of the master transcription factors involved in the stringent regulation of interferon production following virus infection. Up to now, most studies have focuses on the role of IRF-3 in antiviral signaling pathway in autoimmunity, while studies of the IRF-3 transcriptional regulation mechanism are not very clear. Bioinformatic analyses have revealed putative new transcription start sites in intron 1 and 2, indicating that the gene has multiple promoters and spliced isoforms.In the present study, we identified two transcription start sites (TSSs) of IRF-3 by 5` rapid amplification of cDNA ends (5`RACE). DNA sequencing located the TSSs at 718 bp and 162 bp before the first nucleotide of the third exon of IRF-3. Based on their structural features, the corresponding transcripts were named intron 2 variant 1(Int2V1) and intron 2 variant 2(Int2V2). RT-PCR analysis showed that Int2V1 and Int2V2 were expressed differently in different tissues and cells. Progressive deletion analysis from the 5'ends revealed the core promoter region of Int2V1 was located within -159~-100 bp upstream of TSS. Sp1 was confirmed as the key transcription factor in regulating the Int2V1 promoter activity. Child patients with Epstein-Barr virus infection(EBV) tend to have somewhat lower Int2V1 mRNA levels than healthy controls. Int2V1Exposure of transfected 293T cells to viral the double stranded RNA(dsRNA) analogue Poly(I:C) and double stranded DNA(dsDNA) analogue poly (dA:dT) produced a 1.3-4 folds increase in promoter activities of Int2V1. Using RT-PCR, we furthermore show that with prolonged treatment time with Poly(I:C), the Int2V1 mRNA expression was significantly down-regulated. In the poly(dA:dT) group, Int2V1 mRNA expressions increased to reach a peak value in 6-8 hours followed by a decrease over the next 16-18 hours.Our study extends the information on IRF-3 promoter and splice isoform production and enhances our understanding of the transcriptional regulation of IRF-3.