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. 2022 Dec 12;11:e81086. doi: 10.7554/eLife.81086

Figure 1. Insertion of a resistance cassette at the DBP1 locus causes aberrant transcription and reduced protein production from the neighboring MRP51 gene.

(A) Translation (ribosome profiling, footprint) and (B) mRNA abundance (mRNA-seq) reads per kilobase million mapped reads (RPKM) for every ORF expressed in wild-type and dbp1Δ::kanMX6 cells is plotted. (C) Fold-change of translation efficiency (TE: FP RPKM/mRNA RPKM) for all expressed genes. (A–C) Data represent RPKM values from a single experiment, for RPKM values for all quantified genes, see Figure 1—source data 1. (D) Quantification of Mrp51 levels in wild-type and dbp1∆::kanMX6 cells undergoing mitotic growth as determined by western blotting. Mrp51 levels were normalized to alpha tubulin and three independent biological replicates were quantified. Statistical significance was determined by a ratio paired t-test with a reported two-tailed p < 0.05. For representative blot see Figure 1—figure supplement 1A. (E) mRNA and FP reads mapped to the DBP1/MRP51 locus in wild-type (gray/black) and dbp1Δ::kanMX6 (purple) cells. Note that MRP51 transcripts are 5′ extended in the dbp1Δ::kanMX6 cells compared to wild-type (see inset mRNA tracks). The extended transcript contains three AUG-initiated upstream ORFs (uORFs) translated at the expense of the MRP51 ORF (see FP tracks and inset). (F) Model: replacement of DBP1 ORF with a resistance cassette causes aberrant expression of a long undecoded transcript isoform (LUTI) for MRP51, which results in lower Mrp51 protein expression as an off-target effect.

Figure 1—source data 1. RPKM values for mRNA-seq and ribosome profiling data for all genes quantified in the experiment shown in Figure 1 plots.
elife-81086-fig1-data1.xlsx (547.9KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. A published dataset confirms aberrant transcription and mis-regulation of MRP51 following cassette-mediated DBP1 replacement.

Figure 1—figure supplement 1.

(A) Representative western blot demonstrating wild-type and dbp1∆::kanMX6 levels of Mrp51, with replicates quantified in Figure 1D. For uncropped blot image see Figure 1—figure supplement 1—source data 1. (B–E) Ribosome profiling and mRNA-seq of dbp1Δ::hphMX4 and wild-type S288C budding yeast during mitotic growth from Sen et al., 2019. (A) Translation (FP RPKM) and (B) mRNA RPKM counts of all genes expressed. (C) Fold-change TE (translation efficiency; FP RPKM/mRNA RPKM) for every gene expressed in dbp1Δ::hphMX4 and wild-type cells. (D) mRNA and FP reads aligned to the DBP1/MRP51 loci for wild-type (gray/black) and dbp1Δ::hphMX4 (purple). Note the MRP51 mRNA is 5′ extended in the dbp1Δ::hphMX4 cells (mRNA tracks) and the translation of upstream ORFs (uORFs) not present on the wild-type mRNA but MRP51 ORF translation is higher in wild-type cells (FP tracks).
Figure 1—figure supplement 1—source data 1. Zipped folder containing uncropped tif image of western blot shown in Figure 1 with and without labels on the relevant samples and bands.