The NMD-sensitivity of XUT0741 depends on its long 3′ UTR. (A) Schematic representation of the native and mutant alleles of XUT0741. The transcript and the coding region are represented as a red line and a blue arrow, respectively. The sequence of the smORF in the native XUT0741 is indicated. The length of the coding region and of the 3′ UTR is shown beside each allele. The chimera construct was obtained by combining the 5′ UTR and coding region of the xut0741-d allele to the long 3′ UTR of the native XUT0741. (B) WT and upf1Δ cells expressing the different alleles of XUT0741 were grown to mid-log phase, at 30°C, in YPD medium. After total RNA extraction, the levels of each transcript were assessed by strand-specific RT-qPCR, and then normalized on scR1. The gray bars correspond to the levels of the different alleles of XUT0741 in WT cells (native XUT set to 1, indicated by the dashed line). (C) NMD-sensitivity of each allele of XUT0741, given by the ratio between the mean levels of each transcript in the upf1 mutant and the WT strain (see Supplemental Fig. S5B). Mean and SD values were calculated from three independent biological replicates. (**) P < 0.01; (***) P < 0.001; (ns) not significant upon t-test. The dashed line indicates a upf1Δ/WT ratio = 1 (i.e., no NMD-sensitivity). (D) WT and upf1Δ cells expressing the native XUT0741, the xut0741-d allele, and the chimera were grown as described above. Total RNA was extracted and analyzed by northern blot. The different alleles of XUT0741 and scR1 (loading control) were detected using 32P-labeled AMO3581 and AMO1482 oligonucleotides, respectively. (E) NMD-sensitivity of each allele, calculated from northern blot signals in WT and upf1Δ cells (see Supplemental Fig. S5D). Mean and SD values were calculated from three independent biological replicates. (***) P < 0.001; (ns) not significant upon t-test. Dashed line: upf1Δ/WT ratio = 1.
