Talk abstracts
Talk on Saturday 11:00-11:15am submitted by Jacob Smith
Measuring the Transcriptional and Splicing Effects of Optically Activated IRE1 and the Unfolded Protein Response
Jacob W. Smith (Department of Chemistry and Biochemistry, The Ohio State University), 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. IRE1 responds to ER stress by forming homo-oligomers that enable trans-autophosphorylation and activation of the cytosolic RNase domain, which then facilitates non-canonical splicing of the mRNA of transcription factor XBP1 into an active isoform that induces a sweeping transcriptional program. Previous studies have also indicated that IRE1 directly degrades a broad set of RNAs. IRE1 has been shown to play an important role in diseases where cells are faced with chronic ER stress, such as neurodegeneration, diabetes, and several types of cancer. A lack of direct experimental means of activation has made it difficult to study IRE1 signaling independently of the other UPR sensors, leading us to engineer an IRE1 construct (called Opto-IRE1) with an added light-inducible oligomerizing domain to cluster and subsequently activate the cytosolic kinase and RNase domains. By leveraging long-read RNA sequencing, we were able to measure the transcriptional changes induced by Opto-IRE1 and general ER stress. We also used siRNA to knockdown XBP1 and isolate the direct effects of Opto-IRE1 activity, and we found that XBP1-independent changes constitute a small fraction of regulated genes. We also found 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 the splicing changes induced by direct inhibition of NMD. This indicates that the UPR is using an additional mechanism to affect splicing. By leveraging the selectivity of Opto-IRE1 and power of long-read RNA sequencing, our data clarify the effects of IRE1 and the UPR on transcriptional and splicing regulation.
Keywords: Transcriptomics, Splicing, Stress