Albanese, A.; Daly, L. A.; Prost-Fingerle, K.; Elliott, L. G.; Oswald, S. O.; Ecclestone, G. B.; Bell, G.; Batie, M.; Rigden, D. J.; Eyers, C.; Fandrey, J.; Kenneth, N. S.; See, V. S.; Rocha, S.

Oxygen sensing and adaptation are vital for metazoan survival. At the cellular level the response to hypoxia is characterised by a switch in transcriptional programming, primarily mediated by hypoxia-inducible factors (HIFs). HIFs comprise an obligatory heterodimer between an oxygen-sensitive HIF-alpha subunit and an oxygen-insensitive HIF-1beta subunit, which together regulate gene expression in response to hypoxia. Among the three HIF-alpha proteins in vertebrates (HIF-1alpha, HIF-2alpha, and HIF-3alpha), post-translational modifications (PTMs) play a crucial role in modulating their stability, localisation, and activity. While many modifications have been investigated on HIF-1alpha, our knowledge of HIF-2alpharegulation is very limited. Here, we investigated the function of the recently identified HIF-2alpha S345 phosphorylation site and demonstrate that this disrupts the interaction between HIF-2alpha and HIF-1beta, thereby silencing the HIF-2 transcriptional response. Additionally, our findings suggest that this modification redirects HIF-2alpha from its normal transcription factor function towards a role in mRNA fate control, as indicated by mass spectrometry interactome analysis and RNA-immunoprecipitation. Our discoveries highlight a new regulatory mechanism of HIF-2 activity, where S345 phosphorylation impedes HIF-2alpha/HIF-1beta heterodimer formation, creating a switch in HIF-2alpha function. As HIF-2alpha is the only HIF-alpha isoform with a clinically approved inhibitor, these insights are crucial for advancing therapeutic strategies targeting hypoxia signalling.