HIFs enhance the transcriptional activation and splicing of adrenomedullin.

UNLABELLED: Adrenomedullin (ADM) is important for tumor angiogenesis, tumor cell growth, and survival. Under normoxic conditions, the ADM gene was found to produce two alternative transcripts, a fully spliced transcript that produces AM and PAMP peptides and intron-3-retaining transcript that produces a less functionally significant PAMP peptide only. ADM is a well-established hypoxia inducible gene; however, it is not clear which ADM isoform is induced by hypoxia. In this study, it was determined that various cancer and normal cells express two predominant types of ADM transcripts, a AM/PAMP peptide producing full-length transcript in which all introns are removed, and a nonprotein producing I1-3 transcript in which all introns are retained. Interestingly, hypoxia preferentially induced the full-length isoform. Moreover, hypoxia-inducible factors (HIF), but not hypoxia per se, are necessary and sufficient to increase splicing of ADM pre-mRNA. ADM splicing reporters confirmed that transcriptional activation by HIF or other transcription factors is sufficient to enhance splicing. However, HIFs are more potent in enhancing ADM pre-mRNA splicing than other transcriptional activators. Thus, ADM intron retention is not a consequence of abnormal splicing, but is an important mechanism to regulate ADM expression. These results demonstrate a novel function of HIFs in regulating ADM expression by enhancing its pre-mRNA splicing. Importantly, using endogenous and cloned ADM gene, further evidence is provided for the coupling of transcription and RNA splicing.

IMPLICATIONS: Here, a novel function of HIFs in regulating ADM gene expression is identified by enhancing ADM pre-mRNA splicing.