Characterization of a novel transcript of prostaglandin endoperoxide H synthase 1 with a tissue-specific profile of expression

The enzyme prostaglandin endoperoxide H synthase (PGHS) has a pivotal role in the prostanoid biosynthetic pathway because it catalyses the formation o f prostaglandin H, (PGH,), the common precursor of prostanoids. Two PGHS is oforms have been reported, PGHS-1 and PGHS-2, which have 61% identity (at t he amino acid level) and 73% similarity (at the nucleotide level) between t he two human enzymes. Transcription of the PGHS-1 gene leads to the formati on of two transcripts (2.8 and 5.1 kb); two transcripts of 2.8 and 4.5 kb a re produced from the PGHS-2 gene. By Northern blot analysis with the entire coding region of human PGHS-1, 2.8 and 5.1 kb transcripts as well as a nov el 4.5 kb transcript were detected in the human megakaryoblastic cell line MEG-01. We designed a strategy to characterize the 4.5 kb PGHS transcript. Probes specific for each PGHS-1 and PGHS-2 were designed on the basis of th e 3' untranslated region (3' UTR), where no similarity is present. The 4.5 kb transcript was detected only with the PGHS-1-specific 3' UTR probes and not with the PGHS-2-specific 3' UTR probe. To investigate the origin of the 4.5 kb PGHS-1 transcript, the remaining 947 bp of the 5.1 kb PGHS-1 transc ript was generated by 3' rapid amplification of cDNA ends (3' RACE) and seq uenced. A non-canonical polyadenylation signal (AAGAAA) located upstream of a potential cleavage site (CA) was found and could generate the 4.5 kb PGH S-1 transcript. Analysis of the sequence also produced several possible G/U -rich elements downstream of the potential cleavage site. An RNA dot-blot w ith 50 different human tissues was probed with the 4.5 and 5.1 kb PGHS-1-sp ecific probes. A signal for the 4.5 kb PGHS-1 transcript was detected in th e bladder and appendix. Signals of lower intensity were detected in the col on, bone marrow, small intestine, uterus, prostate, peripheral leucocyte, l ymph node and stomach. In conclusion, our results suggest that the cell lin e MEG-01, the bladder and the appendix contain a new PGHS-1 transcript of 4 .5 kb that can be produced from the PGHS-1 gene and we provide a better str ategy for distinguishing PGHS-1 transcripts from PGHS-2.