Poster abstracts
Poster number 33 submitted by Swapnil Mukherjee
Dissecting the mechanism of regulation of an unconventional RNA splicing reaction
Swapnil Mukherjee (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Vladislav Belyy (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
Inositol-requiring enzyme 1 (IRE1) is an ER-localized bifunctional kinase/RNase membrane receptor that primarily senses ER stress. When activated, mammalian IRE1 cleaves a specific mRNA encoding the transcription factor X-box binding protein (XBP1) at two sites which are then ligated by the tRNA ligase RTCB. This unconventional splicing reaction is a key step in ER stress alleviation. The precise mechanism by which oligomerization activates IRE1 is still unknown, in part due to the substantial experimental challenges associated with studying subtle oligomeric transitions in low-affinity protein clusters. To overcome this problem, we engineered a platform to study the consequence of oligomerization on the activity of IRE1. This system comprises the cytosolic domain of mammalian IRE1 fused to components of two orthogonal heterodimerization systems enabling the assembly of dimeric and tetrameric IRE1 oligomers at physiologically relevant concentrations. Phosphorylation or dephosphorylation of the protomers allows us to assemble oligomers of well-defined phosphorylation states. We used in vitro RNA cleavage as a readout to study how the activity of IRE1 is modulated by change in protein state. In agreement with previous reports, we find that the activity of IRE1 increases on dimerization and phosphorylation. However, we do not see any noticeable increase in RNase activity on tetramerization suggesting that dimerization of IRE1 is sufficient for inducing XBP1 cleavage. Surprisingly, our initial results suggest that while phosphorylated monomers exhibit higher activity than dephosphorylated monomers, dimeric forms of IRE1 exhibits similar rates of XBP1 cleavage, regardless of phosphorylation state. This suggests that the role of phosphorylation might be to primarily stabilize the active dimeric state of IRE1. Our results build towards a comprehensive model of how IRE1-dependent signaling is modulated in cells.
Keywords: RNase , Kinase, ER Stress