Protomer alignment modulates specificity of RNA substrate recognition by Ire1

  1. Weihan Li
  2. Kelly Crotty
  3. Diego Garrido Ruiz
  4. Mark Voorhies
  5. Carlos Rivera
  6. Anita Sil
  7. R Dyche Mullins
  8. Matthew P Jacobson
  9. Jirka Peschek  Is a corresponding author
  10. Peter Walter  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Yale School of Medicine, United States
  3. Howard Hughes Medical Institute, University of California, San Francisco, United States

Abstract

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs—non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (W. Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Weihan Li

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4718-1884
  2. Kelly Crotty

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Diego Garrido Ruiz

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2441-385X
  4. Mark Voorhies

    Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8815-7384
  5. Carlos Rivera

    Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Anita Sil

    Microbiology & Immunology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. R Dyche Mullins

    Microbiology & Immunology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Matthew P Jacobson

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Jirka Peschek

    Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    jirka@walterlab.ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8158-9301
  10. Peter Walter

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    peter@walterlab.ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6849-708X

Funding

Howard Hughes Medical Institute

  • R Dyche Mullins
  • Peter Walter

Deutsche Forschungsgemeinschaft (Emmy Noether fellow)

  • Jirka Peschek

UCSF-Zaffaroni Fellowship

  • Weihan Li

Human Frontier Science Program

  • Jirka Peschek

National Institute of Allergy and Infectious Diseases (2R37 AI066224)

  • Anita Sil

National Institute of General Medical Sciences (R01 GM032384)

  • Peter Walter

National Institute of General Medical Sciences (1R35 GM118119-01)

  • R Dyche Mullins

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,146
    views
  • 215
    downloads
  • 8
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Weihan Li
  2. Kelly Crotty
  3. Diego Garrido Ruiz
  4. Mark Voorhies
  5. Carlos Rivera
  6. Anita Sil
  7. R Dyche Mullins
  8. Matthew P Jacobson
  9. Jirka Peschek
  10. Peter Walter
(2021)
Protomer alignment modulates specificity of RNA substrate recognition by Ire1
eLife 10:e67425.
https://doi.org/10.7554/eLife.67425

Share this article

https://doi.org/10.7554/eLife.67425

Further reading

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics
    Kira Breunig, Xuifen Lei ... Luiz O Penalva
    Research Article

    RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. Serpine1 mRNA-binding protein 1 (SERBP1) is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We defined SERBP1’s interactome, uncovered novel roles in splicing, cell division and ribosomal biogenesis, and showed its participation in pathological stress granules and Tau aggregates in Alzheimer’s brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.

    1. Biochemistry and Chemical Biology
    Parnian Arafi, Sujan Devkota ... Michael S Wolfe
    Research Article

    Missense mutations in the amyloid precursor protein (APP) and presenilin-1 (PSEN1) cause early-onset familial Alzheimer’s disease (FAD) and alter proteolytic production of secreted 38-to-43-residue amyloid β-peptides (Aβ) by the PSEN1-containing γ-secretase complex, ostensibly supporting the amyloid hypothesis of pathogenesis. However, proteolysis of APP substrate by γ-secretase is processive, involving initial endoproteolysis to produce long Aβ peptides of 48 or 49 residues followed by carboxypeptidase trimming in mostly tripeptide increments. We recently reported evidence that FAD mutations in APP and PSEN1 cause deficiencies in early steps in processive proteolysis of APP substrate C99 and that this results from stalled γ-secretase enzyme-substrate and/or enzyme-intermediate complexes. These stalled complexes triggered synaptic degeneration in a Caenorhabditis elegans model of FAD independently of Aβ production. Here, we conducted full quantitative analysis of all proteolytic events on APP substrate by γ-secretase with six additional PSEN1 FAD mutations and found that all six are deficient in multiple processing steps. However, only one of these (F386S) was deficient in certain trimming steps but not in endoproteolysis. Fluorescence lifetime imaging microscopy in intact cells revealed that all six PSEN1 FAD mutations lead to stalled γ-secretase enzyme-substrate/intermediate complexes. The F386S mutation, however, does so only in Aβ-rich regions of the cells, not in C99-rich regions, consistent with the deficiencies of this mutant enzyme only in trimming of Aβ intermediates. These findings provide further evidence that FAD mutations lead to stalled and stabilized γ-secretase enzyme-substrate and/or enzyme-intermediate complexes and are consistent with the stalled process rather than the products of γ-secretase proteolysis as the pathogenic trigger.