Poster abstracts
Poster number 47 submitted by Jacob Smith
Probing Transcriptional and Splicing Regulation in the Unfolded Protein Response with Optically Activated IRE1
Jacob W. Smith (Ohio State Biochemistry Program), Vladislav Belyy (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Inositol-Requiring Enzyme 1 (IRE1) is one of three known membrane-resident stress sensors that respond to imbalances in the Endoplasmic Reticulum (ER) by activating the Unfolded Protein Response (UPR), a eukaryotic stress response pathway. Human IRE1 is an essential protein in both development and homeostasis that responds to ER stress by forming transient homo-oligomers. These oligomers enable trans-autophosphorylation and activation of the cytosolic RNase domain, which then initiates non-canonical splicing of the mRNA of transcription factor XBP1 into an active isoform that induces a sweeping transcriptional program. A lack of direct means of activating IRE1 has made it difficult to study independently of the other UPR sensors, leading us to engineer an IRE1 construct (called Opto-IRE1) with a light-inducible oligomerizing domain to cluster and activate the cytosolic kinase and RNase domains. To clarify the role of IRE1 within the UPR, we measured the transcriptional changes induced by Opto-IRE1 and by general ER stress in cultured human cells. With siRNA knockdown of XBP1, we also isolated the direct effects of IRE1 activity and found that XBP1-independent changes constitute a small fraction of IRE1 effects. We also created a reference-free, de novo method for identifying changes in splicing from long-read transcriptomics data. This revealed that general ER stress induced numerous changes in RNA splicing with a strong preference towards intron retention and that these changes were not IRE1-dependent. Although the UPR has been shown to regulate Nonsense-Mediated Decay (NMD), the ER stress-induced splicing changes only partially overlapped with direct inhibition of NMD. This suggests that the UPR is using another mechanism to affect splicing. By leveraging the selectivity of Opto-IRE1, our data reveal the effects of IRE1 and the UPR on transcriptional and splicing regulation.
Keywords: Transcriptomics, Splicing, Stress