Upon environmental or chemical stress, unfolded proteins start accumulating in the endoplasmic reticulum (ER), triggering a specific response to restore homeostasis. This is mediated by three sensors: IRE-1, PERK and ATF-6. The activation of IRE-1 induces recognition of
xbp-1 mRNA and excision of a 23nt sequence via a unique, non-canonical, splicing event. This creates a frameshift in
xbp-1 coding sequence which leads to generation of a protein variant (XBP-1S) that can activate expression of genes involved in the unfolded protein response (UPR). This pathway is strongly associated with neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Studies performed in Caenorhabditis elegans showed an acceleration of neurodegeneration in worms lacking either
ire-1 or
xbp-1. RT-PCR and northern blot analyses generally show that only a small fraction of
xbp-1 mRNAs is spliced in response to ER stress but an RNAseq analysis performed on a
rtcb-1 mutant (the ligase involved in
xbp-1 splicing) seems to indicate that most mRNAs are actually in a spliced state under ER stress in this context (Kosmaczewski et al., 2014). Most studies to date have used RNA extracts to follow the status of
xbp-1 messenger. In this study, we aim to characterize the expression of both XBP-1 isoforms in vivo in C. elegans under both normal and stress-induced conditions. To detect both isoforms in vivo, we are using a two-color reporter system called Frame Shift Sensor (FSS), that encodes fluorescent proteins in different reading frames according to the splicing status of the transcript. When we introduced a construct expressing
xbp-1 fused to the FSS as extrachromosomal arrays, we only detected expression in few neuronal cells. Moreover, upon expression of the construct under a muscle-specific promoter, we observed a spontaneous switch between XBP-1U and XBP-1S during larval development, without any induction of stress. In order to get rid of potential artefacts caused by the introduction of numerous copies of our transgenes as extrachromosomal arrays, we are now working on introducing the FSS directly into the genomic copy of
xbp-1 as well as performing single-genomic insertion of various reporter constructs of interest. Expression of both isoforms will then be characterized by a combination of microscopy and flow cytometry. In parallel we are also investigating the unexpected pattern of expression of
xbp-1 using a tissue-specific RT-PCR to assess the splicing status of
xbp-1 mRNA in muscle cells. This will allow us to determine if our observations are the results of transcriptional or post-transcriptional regulation events. Finally, we will screen for new
xbp-1 splicing regulators by using RNAi assays, which will allow identification of every molecular interactions playing a role in regulating
xbp-1 unconventional splicing.