1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Structural insights into the assembly and polyA signal recognition mechanism of the human CPSF complex

  1. Marcello Clerici
  2. Marco Faini
  3. Ruedi Aebersold  Is a corresponding author
  4. Martin Jinek  Is a corresponding author
  1. University of Zurich, Switzerland
  2. Swiss Federal Institute of Technology, Switzerland
Research Article
  • Cited 35
  • Views 2,858
  • Annotations
Cite this article as: eLife 2017;6:e33111 doi: 10.7554/eLife.33111

Abstract

3' polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Marcello Clerici

    Department of Biochemistry, University of Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  2. Marco Faini

    Department of Biology, Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Ruedi Aebersold

    Department of Biology, Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Zurich, Switzerland
    For correspondence
    aebersold@imsb.biol.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
  4. Martin Jinek

    Department of Biochemistry, University of Zurich, Zurich, Switzerland
    For correspondence
    jinek@bioc.uzh.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7601-210X

Funding

European Research Council (ERC-StG-337284)

  • Marcello Clerici
  • Martin Jinek

European Molecular Biology Organization (ALTF-343-2013)

  • Marco Faini

European Research Council (HEALTH-F4-2008-201648)

  • Ruedi Aebersold

European Research Council (233226)

  • Ruedi Aebersold

H2020 European Research Council (670821)

  • Ruedi Aebersold

Innovative Medicines Initiative Joint Undertaking (ULTRA-DD grant no. 115766)

  • Ruedi Aebersold

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

Reviewing Editor

  1. Nick J Proudfoot, University of Oxford, United Kingdom

Publication history

  1. Received: October 26, 2017
  2. Accepted: December 21, 2017
  3. Accepted Manuscript published: December 23, 2017 (version 1)
  4. Version of Record published: January 9, 2018 (version 2)

Copyright

© 2017, Clerici 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

  • 2,858
    Page views
  • 525
    Downloads
  • 35
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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)

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

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

Further reading

    1. Biochemistry and Chemical Biology
    Maria Carmela Filomena et al.
    Research Article

    Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

    1. Biochemistry and Chemical Biology
    Xavier Portillo et al.
    Research Article Updated

    An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, raising the possibility that further improvement of polymerase activity may be achievable.