ИСТИНА

The S. cerevisiae gene deletion collection is widely used for genome-wide annotation of function and study of genetic interactions. However, the standard G418-resistance cassette used to obtain knockout mutants introduces strong regulatory elements into the target genetic loci. Here, using ribosome profiling data, we analyzed transcriptional and translational 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 and changes in protein abundance. 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, and the appearance of upstream AUG codons inhibited translation of the main open reading frame. In some cases, these events caused false genetic interactions of the deleted genes. Our data describe the interactions of the KanMX cassette with neighboring genes and provide mechanistic insights into the molecular mechanisms involved. They also suggest that caution is needed in interpreting the results of deletion screens, especially those using strong regulatory elements. The study was supported by grants RSF 18-14-00291 and RFBR 19-34-51047.