A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

Abstract

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

Proteolytic Activation of Plant Membrane-Bound Transcription Factors

“…Transcripts of the plant bZIP60, mammalian XBP1, and yeast HAC1 are alternatively spliced by IRE1 isoforms that are activated upon ER stress (for instance, tunicamycin treatment) by oligomerization and autophosphorylation. Consequently, the bZIP mRNAs are alternatively spliced, with a premature stop codon to exclude the TMD as a result ( Yoshida et al, 2001 ; Calfon et al, 2002 ; Nagashima et al, 2011 ; Jäger et al, 2012 ; Diwan et al, 2021 ). Moreover, for the plant bZIP17 and bZIP28 and the mammalian SREBPs/ATF6, the accumulation of unfolded proteins is sensed in the luminal TF part and results in the translocation of the TFs from the ER to the Golgi, where they are released by the S2P metalloprotease ( Ye et al, 2000a , b ; Stirling and O’Hare, 2006 ; Liu et al, 2008 ; Tajima et al, 2008 ; Iwata et al, 2017 ).…”

“…However, our knowledge on the activation mechanisms of MB-TFs in plants is limited to that of bZIP17 and bZIP28, and bZIP60 that are regulated through RIP and alternative spicing, respectively, during the unfolded protein response ( Liu et al, 2008 ; Tajima et al, 2008 ; Nagashima et al, 2011 ). Both activation mechanisms are highly conserved in multicellular life ( Sun et al, 2015 ; Diwan et al, 2021 ). Experimental indication for proteolytic activation of MB-TFs is often based on N-terminomics or pharmacological methods with certain protease inhibitors.…”

Development of a GFP biosensor reporter for the unfolded protein response-signaling pathway in plants: incorporation of the bZIP60 intron into the GFP gene

“…Although RIDD has been shown in plants, the identification of substrates and the mechanisms of such degradation are not well understood. 34 This reporter can also be applied to assess the effects of UPR mutants (i.e. in the ERAD pathway) on affecting the IRE1-based splicing pathway.…”

bZIP60 and Bax inhibitor 1 contribute IRE1‐dependent and independent roles to potexvirus infection

“…After 3–4 d, microscopic observations were made using a FV3000 laser scanning confocal system mounted to an IX83 inverted microscope with High Sensitivity GaAsP PMT detectors (Olympus America, Centre Valley, PA, USA). Immunoblotting to detect bZIP60U and bZIP60S was performed according to (Liu et al ., 2019; Diwan et al ., 2021) and details are provided in the Methods S4.…”

“…The mammalian and plant IRE1 ribonucleases each remove a nonconventional intron from the XBP1/bZIP60 mRNAs to produce the functional transcription factor (Deng et al ., 2011). Another ribonuclease function is the promiscuous IRE1‐dependent decay of mRNA (RIDD) which attacks a wide range of RNA substrates, providing a general effect on translation in response to ER stress (Maurel et al ., 2014; Tam et al ., 2014; Diwan et al ., 2021). The interplay between the UPR machinery and autophagy in mammalian cells includes the activation of the c‐Jun N‐terminal kinase pathway by IRE1 kinase activity.…”