A standard knockout procedure alters expression of adjacent loci at the translational level
A standard knockout procedure alters expression of adjacent loci at the translational level
The S. 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 neighbor and the deleted gene, knockout often led to a shift of the transcription start site of the neighboring gene, 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.
Kushchenko Artem S.、Dmitriev Sergey E.、Kulakovskiy Ivan V.、Gladyshev Vadim N.、Edakin Roman O.、Alexandrov Alexander I.、Makeeva Desislava S.、Egorov Artyom A.、Urakov Valeriy N.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||Sirius University of Science and Technology||Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State UniversityBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||Sirius University of Science and Technology||Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University||Engelhardt Institute of Molecular Biology, Russian Academy of SciencesBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||Sirius University of Science and Technology||Vavilov Institute of General Genetics, Russian Academy of Sciences||Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of SciencesDivision of Genetics, Department of Medicine, Brigham and Women?ˉs Hospital and Harvard Medical SchoolBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||Sirius University of Science and Technology||Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State UniversityBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||FRC of Biotechnology of the Russian Academy of Sciences, Bach Institute of BiochemistryBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||Sirius University of Science and TechnologyPhystech School of Biological and Medical Physics, Moscow Institute of Physics and Technology (State University)||Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University||Sirius University of Science and TechnologyFRC of Biotechnology of the Russian Academy of Sciences, Bach Institute of Biochemistry
遗传学分子生物学细胞生物学
Saccharomyces Genome Deletion Project (SGDP)neighboring gene effect (NGE)yeast gene knockout strainsyeast deletion librarygenetic interactionribosome profilingKanMX4 cassette
Kushchenko Artem S.,Dmitriev Sergey E.,Kulakovskiy Ivan V.,Gladyshev Vadim N.,Edakin Roman O.,Alexandrov Alexander I.,Makeeva Desislava S.,Egorov Artyom A.,Urakov Valeriy N..A standard knockout procedure alters expression of adjacent loci at the translational level[EB/OL].(2025-03-28)[2025-06-12].https://www.biorxiv.org/content/10.1101/2021.08.21.457210.点此复制
评论