Abstract
Epitope-tagging by homologous recombination is ubiquitously used to study gene expression, protein localization and function in yeast. This is generally thought to insulate the regulation of gene expression to that mediated by the promoter and coding regions because native 3′ UTR are replaced. Here we show that the 3′ UTRs, CYC1 and ADH1, contain cryptic promoters that generate abundant convergent antisense-transcription in Saccharomyces cerevisiae. Moreover we show that aberrant, truncating 3′ –end formation is often associated with regulated transcription in TAP-tagged strains. Importantly, the steady-state level of both 3′ –truncated and antisense transcription products is locus dependent. Using TAP and GFP-tagged strains we show that the transcriptional state of the gene-of-interest induces changes to 3′ –end formation by alternative polyadenylation and antisense transcription from a universal 3′ UTR. This means that these 3′ UTRs contains plastic features that can be molded to reflect the regulatory architecture of the locus rather than bringing their own regulatory paradigm to the gene-fusions as would be expected. Our work holds a cautionary note for studies utilizing tagged strains for quantitative biology, but also provides a new model for the study of promoter driven rewiring of 3′ –end formation and regulatory non-coding transcription.
Highlights
The plasticity of the Saccharomyces cerevisiae genetic model is beloved by all who have worked with it
We show that the CYC1 3 UTR, ubiquitously utilized in ectopic expression plasmids generates abundant antisense transcripts, and that epitope tagging with the localization tag GFP, at native genomic loci, can be associated with convergent antisense transcription from the ADH1 3 UTR
We show that antisense-transcripts are induced from cryptic promoters in response to controlling upstream promoter decisions. The evidence for this in our data is provided by the universal ADH1 3 UTR that terminates the Tandem Affinity Purification (TAP) and GFP tagged strains used in this study
Summary
The plasticity of the Saccharomyces cerevisiae genetic model is beloved by all who have worked with it. Easy transformation and efficient homologous recombination has meant that researchers working with baker’s yeast were among the first to embrace designer genomic knock-out [1] and knockin [2,3] collections. These features of easy genetic manipulation have been exceptionally powerful tools in the discovery of global –network based trends and specific gene-by-gene dissection of eukaryotic cell biology. Annotated SUTs and CUTs become cyclically expressed with inverse phasing to their overlapping coding transcripts within the cell-cycle [6]
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