The Dynamic of the Splicing of bZIP60 and the Proteins Encoded by the Spliced and Unspliced mRNAs Reveals Some Unique Features during the Activation of UPR in Arabidopsis thaliana

Abstract

The unfolded protein response (UPR) is a signaling pathway that is activated when the workload of the endoplasmic reticulum (ER) is surpassed. IRE1 is a sensor involved in triggering the UPR and plays a key role in the unconventional splicing of an mRNA leading to the formation of a transcription factor that up-regulates the transcription of genes that play a role in restoring the homeostasis in the ER. In plants, bZIP60 is the substrate for IRE1; however, questions such as what is the dynamics of the splicing of bZIP60 and the fate of the proteins encoded by the spliced and unspliced forms of the mRNA, remain unanswered. In the present work, we analyzed the processing of bZIP60 by determining the levels of the spliced form mRNA in plants exposed to different conditions that trigger UPR. The results show that induction of ER stress increases the content of the spliced form of bZIP60 (bZIP60s) reaching a maximum, that depending on the stimuli, varied between 30 min or 2 hrs. In most cases, this was followed by a decrease in the content. In contrast to other eukaryotes, the splicing never occurred to full extent. The content of bZIP60s changed among different organs upon induction of the UPR suggesting that splicing is regulated differentially throughout the plant. In addition, we analyzed the distribution of a GFP-tagged version of bZIP60 when UPR was activated. A good correlation between splicing of bZIP60 and localization of the protein in the nucleus was observed. No fluorescence was observed under basal conditions, but interestingly, the fluorescence was recovered and found to co-localize with an ER marker upon treatment with an inhibitor of the proteasome. Our results indicate that the dynamics of bZIP60, both the mRNA and the protein, are highly dynamic processes which are tissue-specific and stimulus-dependent.

CoSpliceNet: a framework for co-splicing network inference from transcriptomics data

“…The expression of a previously unknown IRE1A SV, an ER-resident transmembrane protein which carries out unconventional, extra-nuclear, splicing in the cytosol [36, 37], was found to be significantly correlated with the expression of 37 SVs (Additional file 16: Table S13, Fig. 7a).…”

In Brachypodium a complex signaling is actuated to protect cells from proteotoxic stress and facilitate seed filling

“…One possible explanation for the accumulation of unspliced BdbZIP60 in ER stress conditions in Brachypodium is that ER stress may induce BdbZIP60 transcription to levels that overcome the RNase activity of IRE1 to lead to a translated form of BdbZIP60 with specific roles in stress responses. For example, it has been suggested that the unspliced form of AtbZIP60 in Arabidopsis may have a role in salt-stress response (Henriquez-Valencia et al 2015; Parra-Rojas et al 2015). Analogously it is possible that the Brachypodium UPR also relies on the translated unspliced BdbZIP60.…”

Maintaining the factory: the roles of the unfolded protein response in cellular homeostasis in plants

“…Indeed, a close examination of the ER molecular phenotypes associated with bzip60 knockouts and expression patterns of bZIP60 yields some interesting information. The unspliced version of bZIP60 encodes a membrane-bound transcription factor, and while it is found to be transcribed under normal conditions, the accumulation of the protein encoded by unspliced bZIP60 has been debated (Iwata et al, 2008(Iwata et al, , 2009Parra-Rojas et al, 2015). Though methods of detection between studies have been different, it has been shown that the product of the unspliced bZIP60 isoform accumulates to higher levels in seedlings treated with a proteasome inhibitor compared with untreated seedlings (Parra-Rojas et al, 2015).…”

“…The unspliced version of bZIP60 encodes a membrane-bound transcription factor, and while it is found to be transcribed under normal conditions, the accumulation of the protein encoded by unspliced bZIP60 has been debated (Iwata et al, 2008(Iwata et al, , 2009Parra-Rojas et al, 2015). Though methods of detection between studies have been different, it has been shown that the product of the unspliced bZIP60 isoform accumulates to higher levels in seedlings treated with a proteasome inhibitor compared with untreated seedlings (Parra-Rojas et al, 2015). When this factor is taken into context with the observation that a cellular recycling process known as macroautophagy (hereafter termed autophagy) is constitutively active in the bzip60 knockout , it could be hypothesized that the dynamic levels of unspliced bZIP60 products are post-translationally subject to selective protein degradation and may regulate aspects of ER homeostasis, including protection of the ER against autophagy.…”