title: Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro.
abstract: Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1α cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1α generated 2', 3'-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA.
authors.affiliation: Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Sayoko
authors.initials: S
authors.lastname: Shinya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kadokura
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Imagawa
authors.forename: Michihiro
authors.initials: M
authors.lastname: Inoue
authors.forename: Kota
authors.initials: K
authors.lastname: Yanagitani
authors.forename: Kenji
authors.initials: K
authors.lastname: Kohno
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D005091:Exons; D010710:Phosphates / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000302:isolation & purification / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D010710:Phosphates; D012333:RNA, Messenger; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases; D011130:RNA Ligase (ATP)