ИСТИНА

The haploid genome of the budding yeast Saccharomyces cerevisiae contains approximately 6000 genes. Two decades ago, the complete gene knockout library was produced by the Saccharomyces Genome Deletion Project with the goal of assigning functions to genes through phenotypic analyses of the mutants. Each gene was replaced with a KanMX cassette harboring the G418 resistance gene under control of the strong eEF1A promoter. The eEF1A terminator, as well as adjacent vector-derived and artificial tag sequences, were inserted along the gene. Thus, the endogenous genetic loci were substantially modified during this procedure. As yeast has a compact genome with short intergenic regions, introduction of a highly expressed gene module could significantly alter local transcription profiles. However, these alterations have never been quantified. Here, using ribosome profiling and RNA-Seq data for several S.cerevisiae knockout strains, we analyzed transcriptional and translational perturbations induced by the KanMX cassette within the modified genomic loci. In many cases, we discovered significant alterations in gene expression, including severe impairment of translation. These changes could be attributed to shifted transcriptional start sites or activation of alternative polyadenylation signals. The most dramatic changes were observed when a deleted gene was arranged “head-to-head” with the neighboring gene, where a shift of transcription start site of the latter expanded the 5’ untranslated region (UTR), and the appearance of upstream AUG codons, inhibited translation of its main open reading frame. In the “tail to tail” arrangement, activation of alternative polyadenylation signals in the neighboring gene’s transcript and 3’ UTR shortening were found in many cases. In some cases, the dramatic drop in expression level of the neighboring gene agreed with reported genetic interactions of the deleted gene, which can now be viewed as falsely attributed. Our observations report on the interactions of the KanMX cassette with neighboring genes and provide an understanding of the molecular mechanisms involved. They also suggest that caution is needed in interpreting the results of deletion screens. The work was supported by a Russian Federation grant (14.W03.31.0012) and the Ministry of Science and Higher Education of the Russian Federation.