title: Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway.
abstract: Unconventional mRNA splicing on the endoplasmic reticulum (ER) membrane is the sole conserved mechanism in eukaryotes to transmit information regarding misfolded protein accumulation to the nucleus to activate the stress response. In metazoans, the unspliced form of X-box-binding protein 1 (XBP1u) mRNA is recruited to membranes as a ribosome nascent chain (RNC) complex for efficient splicing. We previously reported that both hydrophobic (HR2) and translational pausing regions of XBP1u are important for the recruitment of its own mRNA to membranes. However, its precise location and the molecular mechanism of translocation are unclear. We show that XBP1u-RNC is specifically recruited to the ER membrane in an HR2- and translational pausing-dependent manner by immunostaining, fluorescent recovery after photobleaching, and biochemical analyses. Notably, translational pausing during XBP1u synthesis is indispensable for the recognition of HR2 by the signal recognition particle (SRP), resulting in efficient ER-specific targeting of the complex, similar to secretory protein targeting to the ER. On the ER, the XBP1u nascent chain is transferred from the SRP to the translocon; however, it cannot pass through the translocon or insert into the membrane. Therefore, our results support a noncanonical mechanism by which mRNA substrates are recruited to the ER for unconventional splicing.
authors.affiliation: Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kanda
authors.affiliation: Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan; Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, United Kingdom kkouno@bs.naist.jp yanagi.pausing@gmail.com.
authors.forename: Kota
authors.initials: K
authors.lastname: Yanagitani
authors.affiliation: Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yokota
authors.affiliation: Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Esaki
authors.affiliation: Laboratory of Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan; kkouno@bs.naist.jp yanagi.pausing@gmail.com.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kohno
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008954:Models, Biological; D019913:Nuclear Localization Signals / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D021381:Protein Transport; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism*; D015398:Signal Transduction; D000071717:X-Box Binding Protein 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D019913:Nuclear Localization Signals; D012333:RNA, Messenger; D018271:Signal Recognition Particle; D000071717:X-Box Binding Protein 1
keywords: SRP; XBP1 mRNA; translational pausing; translocon; unfolded protein response