Epitope-tagged yeast strains reveal promoter driven changes to 3′-end formation and convergent antisense-transcription from common 3′ UTRs

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.

Can We Predict Gene Expression by Understanding Proximal Promoter Architecture?

“…At the same time, we note that the strategy of Hurst et al was a heuristic that tried to make the best use of the data already available and did not attempt to model many intricacies, including the cell specificity of ChIP-seq peaks [33], antisense transcription [34,35], temporal dynamics during development [36], long-range promoter-enhancer associations [16], and the existence of chromosomal domains and nuclear subcompartments [37,38]. To model these intricacies would be much more challenging and most likely not possible without new data being generated.…”

Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe

“…After overnight growth, the cell culture was then spread on a 5-FOA plate to select for cells that produce the full-length GFP fusion of the protein of interest ( Fig 1C ). Unlike C-terminal gene tagging with a marker, the scarless C-terminal tagging method does not disrupt the endogenous 3’ UTR, which can be important for mRNA localization [ 13 – 16 , 42 ] and regulation of transcription [ 17 ].…”

“…Sequences in the 3’ UTR can also control the endogenous sub-cellular localization of the mRNA, which is often coupled to the localization and expression of the protein [ 13 – 16 ]. In addition, it was recently shown that the widely used CYC1 and ADH1 3’ UTRs contain cryptic promoters that lead to abundant convergent antisense transcription, which may also interfere with normal protein expression [ 17 ]. Any changes to the UTRs that may affect protein abundances are of concern, especially for quantitative studies that measure protein levels [ 3 , 18 ].…”

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

“…We and others have previously shown that the use of distal polyadenylation sites is linked to upregulation of transcriptional initiation ( Swaminathan and Beilharz, 2016 ) and elongation rate ( Pinto et al, 2011 ). To test the idea that a change in nucleotide metabolism caused by cordycepin treatment leads to an increase in transcription rate and thus use of distal poly(A) sites, 4-thiouracil (4tU) labelling was used.…”