2-Aminopurine Selectively Inhibits Splicing of Tumor Necrosis Factor Alpha mRNA

Abstract

2-Aminopurine (2-AP) inhibits specific kinases that phosphorylate the ␣ subunit of eukaryotic translation initiation factor 2. One of these, PKR, is also involved in signal transduction. We show here that 2-AP selectively inhibits expression of tumor necrosis factor alpha (TNF-␣) mRNA in primary human lymphoid cells. 2-AP does not inhibit transcription of the human TNF-␣ gene, nor does it affect mRNA stability. Instead, the flow of short-lived precursor transcripts into mature TNF-␣ mRNA is blocked. When 2-AP is present during induction, unspliced TNF-␣ precursor transcripts accumulate at the expense of mRNA. Using RNase protection analysis with genomic probes for different exon-intron junctions, we show that 2-AP blocks splicing of TNF-␣ mRNA. Neither the TNF-␤ nor the interleukin-1␤ gene shows such regulation. 2-AP also inhibits splicing of precursor RNA transcribed from an exogenous human TNF-␣ gene. Sequences within this gene thus confer sensitivity to 2-AP. Yet, control is not exerted at a specific splice site. Our results reveal the involvement of a 2-AP-sensitive component, expressed in functional form before induction, in the splicing of TNF-␣ mRNA.

Beryllium-Induced TNF-α Production Is Transcription-Dependent in Chronic Beryllium Disease

“…2AP blocks the splicing of pre-mRNA transcripts. But, as shown previously, TNF-␣ pre-mRNA transcripts are unstable (11). As Be stimulation continues to drive the synthesis of pre-mRNA transcripts, we observed a slight increase in the levels relative to the profound decrease in levels in the presence of PTX.…”

“…Our previous studies show that the percentage of Be-specific CD4 ϩ T cells are found in the range of 1.4-29% in CBD BAL cells (2). Be-induced TNF-␣ pre-mRNA upregulation was inhibited by PTX, a transcription inhibitor (12,13), and the splicing of TNF-␣ pre-mRNA into mature mRNA transcripts was inhibited by 2AP, a splicing inhibitor (11). In addition, the data indicate that this Be-induced TNF-␣ production was associated with Be-specific upregulation of AP-1 and NF-B transcription factors in the nuclei of CBD BAL cells.…”

“…In the present study, we tested the hypothesis that the upregulation of Be-stimulated CBD BAL cell TNF-␣ mRNA and protein was transcription-dependent. To do this, we used pentoxifylline (PTX) to inhibit transcription of the TNF-␣ gene and 2-aminopurine (2AP) to inhibit the splicing of TNF-␣ pre-mRNA into mature mRNA transcripts (11)(12)(13). If Be-induced CBD BAL T cell TNF-␣ production is transcription-dependent, a second goal of our study was determining whether Be exposure upregulates nuclear transcription factors associated with activation of the TNF-␣ gene promoter.…”

“…PTX inhibited the formation of TNF-␣ pre-mRNA transcripts and 2AP inhibited the splicing of these TNF-␣ pre-mRNA transcripts into mature TNF-␣ mRNA transcripts (11)(12)(13). Real-time PCR analysis was performed on treated and untreated control cells after 6 h of Be or LPS exposure.…”

“…Real-time RT-PCR was also used to determine TNF-␣ pre-and mature mRNA levels, using a subset of BAL cells from four subjects with CBD, in the absence and presence of 2AP, a splicing inhibitor (11), or PTX, a transcription inhibitor (12,13). For these experiments, cell concentration was adjusted to 1 ϫ 10 6 cells/ml of complete medium, and 200-l aliquots were then cultured in five wells per treatment.…”