Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation

Abstract

Targeted selection-based-genome-editing approaches in budding yeast have enabled many fundamental discoveries and continue to be used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all phenotypes we detected in cells deleted for DBP1. This behavior resembled the previously observed 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). We demonstrate that divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests its consideration in the design and interpretation of experiments using integrated expression cassettes in other eukaryotes.

The lncRNAMalat1is trafficked to the cytoplasm as a localized mRNA encoding a small peptide in neurons

“…Of these ORFs only M1 showed overlapping ribosome binding and this did not extend equally through the ORF. Similar incomplete coverage has been observed in other short ORF’s (Powers et al 2022; Brar et al 2012). This ribosome association with Malat1 could result in productive translation, serve some regulatory role, or simply be adventitious.…”

Dbp1 is a low performance paralog of RNA helicase Ded1 that drives impaired translation and heat stress response

“…Dbp1 helicase can perform similar functions to Ded1 but it is unclear whether Dbp1 has any unique functional roles or characteristics (Berthelot et al, 2004; Jamieson and Beggs, 1991; Powers et al, 2022; Sen et al, 2019; Weis and Hondele, 2022). In vivo analysis of Dbp1 function has been challenging due to a dearth of conditions in which it is known to be expressed and an unexpected side-effect associated with its deletion via cassette-based genomic replacement.…”

“…This is a result of the inherent bidirectionality of the minimal promoters used in expression cassettes (Powers et al, 2022). This off-target effect results in downregulation of cytosolic translation and was responsible for all mutant phenotypes observed in cells deleted for DBP1 via cassette replacement (Powers et al, 2022).…”

“…We recently showed that this genome editing strategy, which was used to make all published yeast strains deleted for the DBP1 locus (Banroques et al, 2011(Banroques et al, , 2010(Banroques et al, , 2008Berthelot et al, 2004;Sen et al, 2019), causes strong translational down-regulation of DBP1's neighboring ORF, encoding the mitochondrial ribosomal protein Mrp51. This is a result of the inherent bidirectionality of the minimal promoters used in expression cassettes (Powers et al, 2022). This offtarget effect results in downregulation of cytosolic translation and was responsible for all mutant phenotypes observed in cells deleted for DBP1 via cassette replacement (Powers et al, 2022).…”

“…This is a result of the inherent bidirectionality of the minimal promoters used in expression cassettes (Powers et al, 2022). This offtarget effect results in downregulation of cytosolic translation and was responsible for all mutant phenotypes observed in cells deleted for DBP1 via cassette replacement (Powers et al, 2022). One condition in which Dbp1 is highly expressed is meiosis, a highly specialized cell differentiation program that produces haploid gametes from diploid cells.…”