Kinetic competition during the transcription cycle results in stochastic RNA processing

  1. Antoine Coulon
  2. Matthew L Ferguson
  3. Valeria de Turris
  4. Murali Palangat
  5. Carson C Chow
  6. Daniel R Larson  Is a corresponding author
  1. National Institutes of Health, United States
  2. Boise State University, United States
  3. Istituto Italiano di Tecnologia, Italy
  4. National Cancer Institute, National Institutes of Health, United States

Abstract

Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. Here, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release.

Article and author information

Author details

  1. Antoine Coulon

    National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Matthew L Ferguson

    Boise State University, Boise, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Valeria de Turris

    Istituto Italiano di Tecnologia, Rome, Italy
    Competing interests
    The authors declare that no competing interests exist.
  4. Murali Palangat

    National Cancer Institute, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Carson C Chow

    National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Daniel R Larson

    National Cancer Institute, National Institutes of Health, Bethesda, United States
    For correspondence
    dan.larson@nih.gov
    Competing interests
    The authors declare that no competing interests exist.

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 8,689
    views
  • 1,436
    downloads
  • 199
    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. Antoine Coulon
  2. Matthew L Ferguson
  3. Valeria de Turris
  4. Murali Palangat
  5. Carson C Chow
  6. Daniel R Larson
(2014)
Kinetic competition during the transcription cycle results in stochastic RNA processing
eLife 3:e03939.
https://doi.org/10.7554/eLife.03939

Share this article

https://doi.org/10.7554/eLife.03939

Further reading

    1. Cancer Biology
    2. Chromosomes and Gene Expression
    Ashley L Cook, Surojit Sur ... Nicolas Wyhs
    Research Article

    Despite exciting developments in cancer immunotherapy, its broad application is limited by the paucity of targetable antigens on the tumor cell surface. As an intrinsic cellular pathway, nonsense-mediated decay (NMD) conceals neoantigens through the destruction of the RNA products from genes harboring truncating mutations. We developed and conducted a high-throughput screen, based on the ratiometric analysis of transcripts, to identify critical mediators of NMD in human cells. This screen implicated disruption of kinase SMG1’s phosphorylation of UPF1 as a potential disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from human and murine truncating mutations in vitro and murine cells in vivo. Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable human leukocyte antigens (HLA) class I-associated peptides from NMD-downregulated proteins on the surface of human cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression
    Kira A Cozzolino, Lynn Sanford ... Dylan J Taatjes
    Research Article

    Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by Trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS over a 75 min to 24 hr timeframe. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A (CA), via rapid suppression of IFN-responsive transcription factor (TF) activity. We also discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. To further probe Mediator kinase function, we completed cytokine screens and metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways in cell type-specific ways, and broad upregulation of anti-inflammatory lipid mediators occurred specifically in kinase-inhibited cells during hyperactive IFNγ signaling. A subset of these lipids (e.g. oleamide, desmosterol) serve as ligands for nuclear receptors PPAR and LXR, and activation of these receptors occurred specifically during hyperactive IFN signaling in CA-treated cells, revealing mechanistic links between Mediator kinases, lipid metabolism, and nuclear receptor function. Collectively, our results establish CDK8/CDK19 as context-specific metabolic regulators, and reveal that these kinases control gene expression not only via TFs, but also through metabolic changes and splicing. Moreover, we establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.