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. 2014 Apr;34(8):1438–1451. doi: 10.1128/MCB.01584-13

FIG 4.

FIG 4

Edc3-mediated RPS28B mRNA decay requires the Rps28 protein expressed from the RPS28A and RPS28B genes. (A) Deletion of RPS28A leads to an increased steady-state level of RPS28B mRNA. Wild-type (HFY114), edc3Δ (CFY25), rps28aΔ (CFY98), and rps28aΔ edc3Δ (CFY96) cells were grown in YEPD medium at 30°C, and the steady-state levels of RPS28B mRNA in the cells were analyzed by Northern blotting as described in the legend to Fig. 3C. The relative levels of RPS28B mRNAs derived from the Northern blot (left) are depicted in the bar graph (right). The error bars indicate standard deviations of the means of three independent experiments. (B) Deletion of RPS28A decreases the RPS28B mRNA decay rate. Wild-type (HFY114), rps28aΔ (CFY98), and edc3Δ (CFY25) cells were grown in YEPD medium at 30°C. Thiolutin was added to each culture, cells were collected at the indicated time points, and the levels of RPS28B mRNA at each time point were analyzed by Northern blotting, using random-primed probes specific for RPS28B or SCR1 transcripts. (C) Deletion of RPS28B abolishes Edc3-mediated decay of chimeric RPS28B transcripts. A set of RPS28B chimeric alleles was constructed (schematics are shown at the top), and the alleles were individually introduced into EDC3 and edc3Δ cells in RPS28B and rps28bΔ backgrounds (HFY114 and CFY25, and CFY159 and CFY161, respectively). The steady-state levels of the transcript encoded by each of these chimeric alleles in the different strains were analyzed by Northern blotting, with different probes used for hybridization with specific transcripts and strain backgrounds. In the rps28bΔ background, all chimeric transcripts were detected using a random-primed probe specific for the RPS28B transcript. In the RPS28B background, the B-P-B and B-Y-B chimeric mRNAs were detected using random-primed probes specific for the PGK1 and YRA1 transcripts, respectively; the B-B′-B chimeric mRNA was detected using an oligonucleotide probe complementary to its coding region. In the schematic, the RPS28B 3′ UTR regulatory element is indicated by the hatched boxes, and RC denotes reverse complement.