Hypoxia regulates RNA splicing of HIF targets

Hypoxia or reduced oxygen level is frequently observed in solid tumors. Hypoxia plays a significant role in solid tumor progression and metastasis by stabilizing hypoxia inducible factor (HIF) to activate gene transcription. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, it is unclear if HIF promotes tumor progression by increasing a particular RNA isoform or all isoforms of each HIF target gene. To address this question, we analyzed hypoxia response at RNA isoform levels [1, 2]. We report that following HIF activation, the levels of HIF target gene pre-mRNAs are increased; importantly, a particular RNA isoform of HIF target gene is preferentially induced by hypoxia [1, 2]. For example, fully spliced adrenomedullin (ADM) transcripts (ADM FL), not intron1, 2, 3 containing ADM transcripts (ADM I1-3) are increased under hypoxia although both ADM FL and I1-3 isoforms are equally expressed in normoxic cells [2]. Also, pyruvate dehydrogenase kinase 1 full-length (PDK1 FL), not exon-4 skipping isoform (∆E4) is preferentially induced under hypoxia [1]. Although the functional difference between PDK1 FL and ∆E4 protein is not clear, increased production of ADM FL is functionally significant since ADM FL, not ADM I1-3 transcripts code for functional protein. These data indicate that hypoxia regulates HIF target gene expression by regulating two pathways, one to increase the levels of pre-mRNAs of HIF target genes, another to control pre-mRNA splicing of HIF target genes. We also attempt to address the molecular mechanism underlying splicing regulation of HIF target genes. We show that HIF activity not hypoxia per se is necessary and sufficient to alter RNA splicing of HIF target genes such as ADM and PDK1 [1, 2], indicating that transcription factor HIF directly or indirectly controls HIF target gene splicing. Using endogenous and splicing reporter genes, we show that transcription activation of ADM by HIF and any other transcription factors would promote ADM FL RNA isoform [2]. In addition, the ADM gene activation strength determines the efficiency of ADM FL production. These data indicate that intron removal from ADM pre-mRNA is directly regulated by HIF-mediated transcription activation. In contrast, increased PDK1 FL is only observed under condition when endogenous HIF target genes are activated [1]. Additional data indicate that increased levels of PDK1 FL require HIF-mediated activation of splicing factor(s). These data suggest that exon-4 inclusion of PDK1 is indirectly regulated by HIF activity. These results …