1. Chromosomes and Gene Expression
  2. Genetics and Genomics

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
https://doi.org/10.7554/eLife.81086
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Metrics

  • 2,288
    views
  • 229
    downloads
  • 5
    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. 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

Categories and tags

Research organism

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals

    Lisa Baumgartner, Jonathan J Ipsaro ... Julius Brennecke
    Research Advance

    Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino’s chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino’s chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression

    Human DDX6 regulates translation and decay of inefficiently translated mRNAs

    Ramona Weber, Chung-Te Chang
    Research Article

    Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

    1. Chromosomes and Gene Expression

    Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation

    Marwan Anoud, Emmanuelle Delagoutte ... Jean-Paul Concordet
    Research Article

    Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades’ radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.