XUTs accumulate upon translation elongation inhibition, independently of NMD. (A) WT (YAM1) cells were grown to mid-log phase in rich (yeast extract–peptone–dextrose, YPD) medium at 30°C. CHX was then added at a final concentration of 100 μg/mL, and samples were collected at different time points. Untreated xrn1Δ (YAM6) and upf1Δ (YAM202) cells, grown under the same conditions, were used as controls. Total RNA was extracted and analyzed by northern blot. XUT1678, XUT0741, the 5′ ITS1 fragment (as well as the 20S pre-rRNA it derives from), and scR1 (loading control) were detected using 32P-labeled AMO1595, AMO1762, AMO496, and AMO1482 oligonucleotides, respectively. Note that probe AMO1595 also detects SUT768, an overlapping shorter stable isoform of XUT1678, which can be detected in WT cells (Wery et al. 2016). (B) Total RNA-seq was performed using total RNA extracted from exponentially growing WT (YAM1) cells (grown as above) treated for 15 min with CHX (100 μg/mL, final concentration) or with an equal volume of DMSO (control). The scatter plot shows the RNA-seq signals (tag densities, log2 scale) for the NMD-sensitive XUTs, mRNAs (light gray dots), and snoRNAs (black dots) in CHX-treated and control WT cells. Up-regulated (CHX/control fold change [FC] >2, P-value < 0.05) and unaffected NMD-sensitive XUTs are represented as red and dark gray dots, respectively (see also Supplemental Table S1). (C) Total RNA-seq was performed in WT (YAM1) and upf1Δ (YAM202) cells, including or not a treatment with CHX (15 min, 100 μg/mL final concentration) or ANS (30 min, 100 μg/mL final concentration). Densities were computed for NMD-sensitive and NMD-insensitive XUTs, using our previously published annotation (Wery et al. 2016). The sensitivity to NMD and/or CHX/ANS of each transcript is shown as a heatmap of the FC (log2 scale) relative to the corresponding control WT cells (treated for the same time with an equal volume of DMSO). Note in the first column that some XUTs (97) previously annotated as NMD-sensitive here show a fold enrichment <2 in the upf1Δ mutant (see Supplemental Table S1), probably reflecting some variability between independent experiments. (D) Same as above. The data are presented as densities (tag/nt, log2 scale) for NMD-sensitive and NMD-insensitive XUTs in control (DMSO) or CHX-treated WT (YAM1) and upf1Δ (YAM202) cells. (***) P-value < 0.001; (ns) not significant upon two-sided Wilcoxon rank-sum test (adjusted for multiple testing with the Benjamini–Hochberg procedure).
