Acute Endoplasmic Reticulum Stress-Independent Unconventional Splicing of XBP1 mRNA in the Nucleus of Mammalian Cells.

Yuanyuan Wang,Yanfen Cui,Wenwen Wang,Pan Xing,Guoguang Ying,Xin Wang,Binghui Li,Wenjing Cui

doi:10.3390/ijms160613302

Abstract

The regulation of expression of X-box-binding protein-1 (XBP1), a transcriptional factor, involves an unconventional mRNA splicing that removes the 26 nucleotides intron. In contrast to the conventional splicing that exclusively takes place in the nucleus, determining the location of unconventional splicing still remains controversial. This study was designed to examine whether the unconventional spicing of XBP1 mRNA could occur in the nucleus and its possible biological relevance. We use RT-PCR reverse transcription system and the expand high fidelity PCR system to detect spliced XBP1 mRNA, and fraction cells to determine the location of the unconventional splicing of XBP1 mRNA. We employ reporter constructs to show the presence of unconventional splicing machinery in mammal cells independently of acute endoplasmic reticulum (ER) stress. Our results reveal the presence of basal unconventional splicing of XBP1 mRNA in the nucleus that also requires inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α) and can occur independently of acute ER stress. Furthermore, we confirm that acute ER stress induces the splicing of XBP1 mRNA predominantly occurring in the cytoplasm, but it also promotes the splicing in the nucleus. The deletion of 5′-nucleotides in XBP1 mRNA significantly increases its basal unconventional splicing, suggesting that the secondary structure of XBP1 mRNA may determine the location of unconventional splicing. These results suggest that the unconventional splicing of XBP1 mRNA can take place in the nucleus and/or cytoplasm, which possibly depends on the elaborate regulation. The acute ER stress-independent unconventional splicing in the nucleus is most likely required for the maintaining of day-to-day folding protein homeostasis.

Highlights

In higher eukaryotes, the unfolded protein response (UPR)-associated adaptive system includes three different signaling pathways mediated by the endoplasmic reticulum (ER) stress sensors, inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), protein kinase-like ER kinase (PERK), and activation of transcription factor 6 (ATF6) [1,2]
The properly processed 5′-LTR–3′-LTR RNA is exported to the cytoplasm, the reverse complement sequence of ER stress activation indicators (ERAI) mRNA of which cannot be unconventionally spliced but be detected using RT-PCR. These results clearly demonstrated that the basal unconventional splicing of X-box-binding protein-1 (XBP1) mRNA sensor sequence in ERAI occurred in the nucleus independent of acute ER stress, suggesting the existence of the basal unconventional machinery of XBP1 mRNA in the nucleus
Since our results showed that the unconventional splicing machinery in both nucleus and cytoplasm required IRE1α (Figure 2A), we tested whether acute ER stress increased the localization of IRE1α to the nucleus

Summary

Introduction

The unfolded protein response (UPR)-associated adaptive system includes three different signaling pathways mediated by the endoplasmic reticulum (ER) stress sensors, inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), protein kinase-like ER kinase (PERK), and activation of transcription factor 6 (ATF6) [1,2]. Upon ER stress, IRE1α is activated and initiates the unconventional splicing of the mRNA encoding X-box-binding protein-1 (XBP1) [3]. After the 26 nt intron within the open reading frame of the XBP1 mRNA is cleaved by IRE1α, the relegated. In contrast to the conventional splicing that usually is catalyzed by the spliceosome and involves a consensus sequence at the flanks of introns according to the Chambon’s rule [5], the unconventional splicing of XBP1 mRNA is performed by IRE1α and an unknown RNA ligase [6,7]. The 26 nt intron of XBP1 mRNA lacks the Chambon’s consensus sequence at its ends, and locates within a pair of characteristic stem-loop sequences.

Methods

Results

Conclusion