Cytoplasmic IRE1α-mediated XBP1 mRNA Splicing in the Absence of Nuclear Processing and Endoplasmic Reticulum Stress

Sung Hoon Back,Kyungho Lee,Elizabeth Vink,Randal J Kaufman

doi:10.1074/jbc.m602030200

Abstract

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signal transduction program termed the unfolded protein response (UPR). In mammalian cells, the UPR is signaled in part through dimerization of ER membrane-localized IRE1alpha to activate its protein kinase and endoribonuclease activities. Activated IRE1alpha cleaves XBP1 mRNA at two sites to initiate an unconventional splicing reaction. The 5' and 3' fragments are subsequently joined by an RNA ligase activity, thereby removing a 26-base intron. This splicing reaction creates a translational frameshift to produce a functional XBP1 transcription factor. However, the cellular location and physiological processes required for splicing of XBP1 mRNA are not well characterized. To study these processes, XBP1 mRNAs were engineered in which translation of enhanced green fluorescence protein or luciferase required splicing of the XBP1 intron. Using cell lines that continuously or transiently express these reporter constructs, we show that cytoplasmic unspliced XBP1 mRNA is efficiently spliced by activated IRE1alpha and requires ongoing cellular transcription but not active translation. The XBP1 intron was effectively removed from RNA substrates transcribed from T7 RNA polymerase or delivered directly to the cytoplasm by RNA transfection, thus indicating that the splicing reaction does not require nuclear processing of the RNA substrate. Analysis of nuclear and cytoplasmic RNA fractions demonstrated that XBP1 mRNA splicing occurs in the cytoplasm. Moreover, an artificial F(v)-IRE1alphaDeltaN was engineered that was able to splice XBP1 mRNA upon chemical-induced dimerization. These findings demonstrate that IRE1alpha dimerization is sufficient to activate XBP1 mRNA splicing in the absence of the UPR. We propose that XBP1 mRNA cytoplasmic splicing provides a novel mechanism to rapidly induce translation of a transcription factor in response to a specific stimulus.

Highlights

Cell: including calcium storage and gated release, biosynthesis of membrane and secretory proteins, and production of lipids and sterols
There was a low level of mXBP1⌬C(s)-d2EGFP protein expression in the absence of an endoplasmic reticulum (ER) stress-inducing agent, the fluorescence intensity, expression, and splicing of mXBP1⌬C(un)d2EGFP mRNA all significantly increased after Tm treatment
Our findings indicate that mammalian IRE1␣-dependent splicing of X-box binding protein 1 (XBP1) mRNA transcripts displays some of the same properties for HAC1 mRNA splicing characterized in S. cerevisiae

Summary

Introduction

Cell: including calcium storage and gated release, biosynthesis of membrane and secretory proteins, and production of lipids and sterols. This construct was transfected into DHFR-deficient CHO cells, and a cell line was selected as described under “Experimental Procedures.” Because the d2EGFP-fused mXBP1⌬C(s) protein is translated in cells that activate the IRE1␣ signaling pathway, UPR activation can be monitored by analysis of d2EGFP fluorescence in living cells (Fig. 1B). These findings suggest that in Tg-treated cells, Act D treatment does not affect the kinase/RNase activity of IRE1␣ and/or the kinase activity of PERK, but rather reduces the splicing of XBP1 mRNA and inhibits expression of downstream genes through the transcriptional blockade.

Results

Conclusion