Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading

Abstract

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, RNA binding proteins and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.

Data availability

All data has been made freely available. Please see Page 21 of the manuscript for Accession numbers.

The following data sets were generated

Article and author information

Author details

  1. Hayley Porter

    Research Department of Cancer Biology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Yang Li

    Research Department of Cancer Biology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Maria Victoria  Neguembor

    Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1583-1304
  4. Manuel Beltran

    Regulatory Genomics Group, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Wazeer Varsally

    Research Department of Cancer Biology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Laura Martin

    Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8801-6637
  7. Manuel Tavares Cornejo

    Regulatory Genomics Group, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Dubravka Pezic

    Research Department of Cancer Biology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Amandeep Bhamra

    Proteomics Research, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Silvia Surinova

    Proteomics Research, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  11. Richard G Jenner

    Regulatory Genomics Group, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Maria Pia Cosma

    Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4207-5097
  13. Suzana Hadjur

    Research Department of Cancer Biology, University College London, London, United Kingdom
    For correspondence
    s.hadjur@ucl.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3146-3118

Funding

Wellcome Trust (106985/Z/15/Z)

  • Suzana Hadjur

Cancer Research UK (PhD studentship)

  • Hayley Porter

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

Reviewing Editor

  1. Andrés Aguilera, CABIMER, Universidad de Sevilla, Spain

Publication history

  1. Received: April 10, 2022
  2. Accepted: April 2, 2023
  3. Accepted Manuscript published: April 3, 2023 (version 1)

Copyright

© 2023, Porter 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

  • 759
    Page views
  • 242
    Downloads
  • 0
    Citations

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

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. Hayley Porter
  2. Yang Li
  3. Maria Victoria  Neguembor
  4. Manuel Beltran
  5. Wazeer Varsally
  6. Laura Martin
  7. Manuel Tavares Cornejo
  8. Dubravka Pezic
  9. Amandeep Bhamra
  10. Silvia Surinova
  11. Richard G Jenner
  12. Maria Pia Cosma
  13. Suzana Hadjur
(2023)
Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading
eLife 12:e79386.
https://doi.org/10.7554/eLife.79386

Further reading

    1. Cell Biology
    Ignacio Bravo-Plaza, Victor G Tagua ... Miguel A Peñalva
    Research Article

    Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

    1. Cell Biology
    Sandipan Dasgupta, Daniella Y Dayagi ... Jeffrey E Gerst
    Research Article

    Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically, <1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.