The Role of Cytoplasmic Polyadenylation Element Binding Protein 2 Alternative Splicing in Chronic Hypoxia

Abstract

Cytosolic polyadenylation element binding (CPEB) proteins are established as mRNA binding proteins that, upon interacting with a 3 ‘UTR consensus sequence, regulate the translation of their mRNA targets. Moreover, the stress-induced CPEB family member, CPEB2, has been shown to interact with the oxygen-dependent subunit of the HIF1 (hypoxia-inducible factor 1) transcription factor, HIF1a, in order to repress its translation. However, our lab has previously demonstrated in cancer cells that alternatively spliced isoforms of CPEB2 regulate the translation of HIF1a in opposing fashions: the CPEB2A isoform represses its translation, while the CPEB2B isoform activates its translation. Although it is well established that during hypoxia, HIF1α levels are initially upregulated due to a decrease in proteasomal degradation, we hypothesize that during chronic hypoxia, the expression of HIF1α is maintained through a stress-induced translational mechanism, likely alongside a decrease in proteolytic activity. In this study we demonstrate that targeting the CPEB2B splice isoform via antisense oligonucleotides has an inhibitory effect on the translation of nascent HIF1α protein during chronic hypoxia, but not during acute hypoxia exposure, and that this this regulatory pathway is dependent on the initiation factor eIF3H. We further demonstrate that CPEB2A and CPEB2B bind differentially to cytoplasmic polyadenylation element consensus sequences, which may depend on surrounding sequence context that can be detected through RNA foot-printing assays. Finally, we characterize how CPEB2A and B expression impacts new blood vessel growth in a 3D vasculature cell model. These findings are important, since they provide evidence that this splicing event could potentially act as a therapeutic target for treating chronic hypoxia-related pulmonary diseases.