Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation

  1. Emily N Powers
  2. Charlene Chan
  3. Ella Doron-Mandel
  4. Lidia Llacsahuanga Allcca
  5. Jenny Kim Kim
  6. Marko Jovanovic
  7. Gloria A Brar  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Columbia University, United States

Abstract

Targeted selection-based-genome-editing approaches in budding yeast have enabled many fundamental discoveries and continue to be used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all phenotypes we detected in cells deleted for DBP1. This behavior resembled the previously observed 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). We demonstrate that divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests its consideration in the design and interpretation of experiments using integrated expression cassettes in other eukaryotes.

Data availability

mRNA sequencing and ribosome profiling data are available at NCBI GEO, with accession numbers GSE207267 and GSE207189. Mass spectrometry data are available at MassIVE, with accession number MSV000089724

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Emily N Powers

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4497-7318
  2. Charlene Chan

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Ella Doron-Mandel

    Department of Biological Sciences, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Lidia Llacsahuanga Allcca

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Jenny Kim Kim

    Department of Biological Sciences, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Marko Jovanovic

    Department of Biological Sciences, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Gloria A Brar

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    gabrar@berkeley.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8560-9581

Funding

National Institutes of Health (R35GM134886)

  • Gloria A Brar

National Institutes of Health (R35GM128802)

  • Marko Jovanovic

National Science Foundation

  • Emily N Powers

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

Reviewing Editor

  1. L Stirling Churchman, Harvard Medical School, United States

Version history

  1. Received: June 15, 2022
  2. Preprint posted: June 29, 2022 (view preprint)
  3. Accepted: November 28, 2022
  4. Accepted Manuscript published: December 12, 2022 (version 1)
  5. Version of Record published: December 15, 2022 (version 2)

Copyright

© 2022, Powers 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.

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  1. Emily N Powers
  2. Charlene Chan
  3. Ella Doron-Mandel
  4. Lidia Llacsahuanga Allcca
  5. Jenny Kim Kim
  6. Marko Jovanovic
  7. Gloria A Brar
(2022)
Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation
eLife 11:e81086.
https://doi.org/10.7554/eLife.81086

Share this article

https://doi.org/10.7554/eLife.81086

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