4.8 Article

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

Journal

NUCLEIC ACIDS RESEARCH
Volume 49, Issue 19, Pages 11134-11144

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab872

Keywords

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Funding

  1. Russian Foundation for Basic Research [19-34-51047]
  2. Russian Ministry of Science and Higher Education
  3. Russian Foundation for Basic Research grant

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The use of the standard G418-resistance cassette for producing knockout mutants in Saccharomyces cerevisiae has potential side effects on neighboring gene expression, affecting transcription efficiency and mRNA translation. Knockout can lead to changes in the transcription start site or alternative polyadenylation signals of neighboring genes, influencing their 5' and 3' UTRs. These events may explain the phenomenon of false genetic interactions due to neighboring gene effects.
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 similar to 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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