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A standard knockout procedure alters expression of adjacent loci at the translational level

Egorov, Artyom A. LU orcid ; Alexandrov, Alexander I. ; Urakov, Valery N. ; Makeeva, Desislava S. ; Edakin, Roman O. ; Kushchenko, Artem S. ; Gladyshev, Vadim N. ; Kulakovskiy, Ivan V. and Dmitriev, Sergey E. (2021) In Nucleic Acids Research 49(19). p.11134-11144
Abstract

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head'... (More)

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head' orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5′ untranslated region (5′ UTR). In the 'tail-to-tail' arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3′ UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

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author
; ; ; ; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
subject
in
Nucleic Acids Research
volume
49
issue
19
pages
11134 - 11144
publisher
Oxford University Press
external identifiers
  • scopus:85120706458
  • pmid:34606617
ISSN
0305-1048
DOI
10.1093/nar/gkab872
language
English
LU publication?
no
additional info
Publisher Copyright: © 2021 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.
id
988c0d8b-9bb1-4605-ab21-b6d031a871f3
date added to LUP
2022-08-24 23:15:26
date last changed
2024-05-27 04:23:33
@article{988c0d8b-9bb1-4605-ab21-b6d031a871f3,
  abstract     = {{<p>The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head' orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5′ untranslated region (5′ UTR). In the 'tail-to-tail' arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3′ UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (&gt;2-fold) deregulated at the level of translation.</p>}},
  author       = {{Egorov, Artyom A. and Alexandrov, Alexander I. and Urakov, Valery N. and Makeeva, Desislava S. and Edakin, Roman O. and Kushchenko, Artem S. and Gladyshev, Vadim N. and Kulakovskiy, Ivan V. and Dmitriev, Sergey E.}},
  issn         = {{0305-1048}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{19}},
  pages        = {{11134--11144}},
  publisher    = {{Oxford University Press}},
  series       = {{Nucleic Acids Research}},
  title        = {{A standard knockout procedure alters expression of adjacent loci at the translational level}},
  url          = {{http://dx.doi.org/10.1093/nar/gkab872}},
  doi          = {{10.1093/nar/gkab872}},
  volume       = {{49}},
  year         = {{2021}},
}