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Messenger RNA targeting to endoplasmic reticulum stress signalling sites

Abstract

Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR)1,2,3. ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5′ untranslated region4. After excision of the intron, transfer RNA ligase joins the severed exons5,6, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue4. Hac1 in turn drives the UPR gene expression program comprising 7–8% of the yeast genome7 to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3′ untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3′ untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.

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Figure 1: A conserved element in the 3′ UTR of HAC1 mRNA is required for splicing in vivo , but not in vitro.
Figure 3: The HAC1 mRNA/Ire1 foci are functional UPR signalling centres.
Figure 2: In response to ER stress HAC1 mRNA localizes to Ire1 foci in a 3′ BE-dependent manner.
Figure 4: Translational repression is a prerequisite for mRNA targeting to Ire1 foci.

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Acknowledgements

We thank M. Jonikas and B. Kornmann for their help with the MatLab scripts; R. Parker for the pPS2037 and pRP1187 plasmids; K. Thorn for the pKT127 plasmid and for his assistance with microscopy at the Nikon Imaging Center at UCSF; and C. Guthrie, R. Andino, J. Gross and members of the Walter laboratory for discussion and comments on the manuscript. T.A. was supported by the Basque Foundation for Science and the Howard Hughes Medical Institute; E.v.A. by the Netherlands Organization for Scientific Research (NWO); D.P. and C.A.R. by the National Science Foundation; C.A.R. by the President’s Dissertation Year Fellowship; and A.V.K. by the Jane Childs Memorial Fund for Medical Research. P.W. is an Investigator of the Howard Hughes Medical Institute.

Author Contributions T.A. and E.v.A. wrote the manuscript, conceived the experiments and together with D.P. carried out most of the experimental work. I.M.S. and E.v.A. observed Ire1 foci, and C.A.R. performed all experiments concerning tRNA ligase localization. A.V.K. carried out kinetic analyses. P.W. directed the research programme and writing of the manuscript.

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Correspondence to Tomás Aragón.

Supplementary information

Supplementary Information 1

This file contains Supplementary Figures S1-S4 with Legends. (PDF 8494 kb)

Supplementary Information 2

This file contains the script which calculates (1) The fraction of Ire1 concentrated in foci and (2) The colocalization index (CI), that scores for the fraction of RNA foci colocalizing with Ire1. (TXT 2 kb)

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Aragón, T., van Anken, E., Pincus, D. et al. Messenger RNA targeting to endoplasmic reticulum stress signalling sites. Nature 457, 736–740 (2009). https://doi.org/10.1038/nature07641

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