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. 2010 Jul 15;24(14):1491–1495. doi: 10.1101/gad.1930710

Figure 4.

Figure 4.

NMD-regulation of Alr1p levels is attributable to a uORF in the ALR1 5′-UTR. (A) Growth in the absence of lysine. The indicated strains (MJY142, MJY67, MJY363, and MJY365) were grown overnight in SC medium at 30°C, serially diluted, spotted onto SC and SC-lys plates, and incubated for 3 d at 30°C. (B) Northern analysis of total RNA isolated from indicated strains (MJY142, MJY67, MJY363, and MJY365) grown in SC medium at 30°C. The blot was probed for LYS2 and SCR1 transcripts using randomly labeled DNA fragments. (C) The chromosomal region upstream of the ALR1 ORF. uORF1, uORF2A/2B, and uORF3 are shown as boxes. Transcriptional start sites, as determined by 5′ RLM-RACE, are indicated with gray arrows (identified in upf1Δ [MJY67] cells) and black arrows (identified in both wild-type [MJY142] and upf1Δ cells). The dashed arrow indicates the presence of an extended subspecies encompassing uORF1 and uORF2A (see the Supplemental Material). (D) Western analysis of the indicated strains (MJY290, MJY309, MJY422, and MJY424) grown in SC medium at 30°C. uORF3 was eliminated by changing the sequence for the AUG codon to AAG. Monoclonal antibodies against HA or Pgk1p were used to detect the indicated proteins.