Abstract
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
Highlights
5-Lipoxygenase is the key enzyme in the biosynthesis of LTs which represent mediators of inflammatory and allergic reactions that are synthesized and released after cell stimulation [1,2]
Induction of immature and/or alternatively spliced 5-LO transcripts preceded the increase in mature 5-LO mRNA following differentiation of Mono Mac 6 (MM6) cells by calcitriol and transforming growth factor-b (TGFb)
Since nonsense-mediated mRNA decay (NMD) depends on mRNA translation, MM6 cells were treated with the protein biosynthesis inhibitor puromycin. 5-LOD3 mRNA expression was significantly upregulated by puromycin
Summary
5-Lipoxygenase (arachidonate:oxygen 5-oxido-reductase) is the key enzyme in the biosynthesis of LTs which represent mediators of inflammatory and allergic reactions that are synthesized and released after cell stimulation [1,2]. Using Northern blot analysis they have shown that the 5-LO gene is expressed as a multi-transcript family (2.7, 3.1, 6.4 and 8.6 kb) in human brain tumors and in dimethyl sulfoxide-differentiated HL-60 cells. Boudreau et al described two novel 5-LO protein isoforms in human leukocytes that are generated from transcripts lacking exon 13 and parts of exon 10, respectively. Both isoforms are catalytically inactive and inhibit the biosynthetic capacity of wild type (WT) 5-LO in cells. Gene expression can be regulated posttranscriptionally by generating alternatively spliced transcripts which are degraded by NMD but can rapidly switch to productive splicing by changing the splicing pattern. We have identified novel splice variants of 5-LO in TGFb/calcitriol differentiated and undifferentiated MM6
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