A03: The roles of architectural IncRNAs in the splicing response to hypoxia

Architectural RNAs (arcRNAs) play key roles in nuclear organisation. Through multivalent interactions with RNA-binding proteins (RBPs), they form nuclear condensates that compartmentalise the nucleus and regulate nuclear functions. Due to their dynamic appearance and the reversible sequestration of specific RBPs, nuclear condensates have the potential to regulate the global reprogramming of gene expression in response to stress, including hypoxia. In the first funding period, we discovered that hypoxia dissolves nuclear speckles (NS), nuclear condensates that store splicing factors. NS dispersal leads to the release of splicing factors, causing global exon skipping, which is essential for hypoxia adaptation. We showed that NS dispersal requires low levels of SRSF6 and uncovered the mechanism for its reduction. To find new types of condensates that form or dissolve in response to hypoxia, we established an experimental workflow and a computational pipeline to identify semi-extractable arcRNAs from RNA-seq data. We focussed on a previously unknown 5’ extension of the microRNA (miRNA) host gene MIR503HG, which we identified as a novel arcRNA candidate in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia. The host gene MIR503HG is linked to endothelial-to-mesenchymal transition in vascular disease. The MIR503HG 5’extension is specifically expressed in endothelial cells, where it binds the RBP FUBP1 and assembles nuclear foci that strongly increase in hypoxia and localise to the phase border of NS. Our pipeline also revealed that many mRNAs retain ultra-conserved introns that are semiextractable and likely convert the affected mRNAs into arcRNAs that perform coding-independent functions. Some introns are exclusively retained in hypoxia, but their role in nuclear organisation has not been explored. As a proof-of-principle, we could already show that intron 3 of SRSF7, which is more retained in hypoxia and semi-extractable, forms large nuclear condensates that auto-regulate the levels of SRSF7. In the second funding period, we will unravel the roles of the newly identified arcRNAs and the dynamics of nuclear condensates in the hypoxia response in endothelial cells. Our experiments in HUVECs revealed a collection of promising arcRNA candidates that will be the focus in the second funding period. For example, in preliminary data, we found that two retained introns (6+7) in the FUS transcript are semi-extractable, densely bound by the RBPs FUS, TAF15 and TDP43 and assemble FUS bodies, which disperse upon hypoxia. Following up on these observations, we will combine cell biology, biochemistry and high-resolution microscopy with computational analyses and multimodal data integration to (i) decipher the non-coding functions of semiextractable intron retention transcripts in nuclear organisation, (ii) investigate the mechanisms and the impact of nuclear condensate assembly or dispersal during hypoxia adaptation, and (iii) study the regulatory role and physiological relevance of arcRNAs in the alternative splicing response in hypoxia for endothelial cells.