Supplementary Figure 5: The nop53 5Ă—Ala mutant is genetically and functionally linked to cytoplasmic surveillance factors.
From: Preribosomes escaping from the nucleus are caught during translation by cytoplasmic quality control
(a) Growth analysis comparing a wild-type strain and the indicated deletions strains under galactose overexpression of plasmid-based NOP53 wild-type or nop53 5×Ala mutant alleles (under control of the GAL1-10 promoter). An empty vector served as control. Cells were spotted in 10-fold serial dilutions on SDC-Leu (glucose) and SGC-Leu (galactose) medium and cell growth at 30°C was monitored after 2 and 3 days, respectively. (b) Polysome gradient analysis of whole cell lysates derived from a Nsa3-Flag and Nsa3-Flag ski2Δ strain after overexpression (for 8 h) of the dominant negative GAL::nop53 5×Ala mutant. The fractions containing 40S, 60S, 80S and polysomes are indicated. The sucrose gradient fractions were analyzed by western blot analysis and probed with the indicated antibodies. (c) Analysis of pre-rRNA and rRNAs extracted from the indicated yeast strains. Galactose induction was performed for 8h, following which RNAs were extracted and analyzed by Northern blotting. Oligonucleotide probes used for Northern analysis: precursors to 5.8S rRNA (top panel) were detected with the 020 oligo probe (TGAGAAGGAAATGACGCT) and the 5S rRNA was detected with the 041 oligo probe (CTACTCGGTCAGGCTC). Values shown indicate the relative abundance of 7S pre-rRNA compared to the wild-type lane (1), when normalized to 5S rRNA levels. Asterisk marks the abnormal processing intermediate between 7S and 5.8S+30.
