In vivo pulse-chase and proteomic analyses of the pre-rRNA processing in exponential and postdiauxic shift cultures.

<p>(A) Low density (OD₆₀₀ = 2) YMK118 was pulse labeled in vivo with ³H-uracil for 6 min and then chased with nonradioactive uracil for 10 min. (B) Yeast culture in early postdiauxic phase (OD₆₀₀ = 10) was pulse labeled for 6 min. The culture was split, and one-half was chased for 10 min, as in (A) (on the left). To the other half of the culture, fresh media and no-radioactive uracil was added to the same final concentration and chased for 10 min (right side, “exponential chase”). (C) Comparison of intensity based absolute quantification (iBAQ) values normalized to bait of 324 proteins detected in the affinity purified preribosomes from pre- and postdiauxic cultures. Proteins changed more than 2-fold are in black. (D) Histogram of SILAC Heavy/Light (H/L) ratios (normalized to bait) for proteins with more than 2-fold change in the preribosomes purified from postdiauxic shift versus prediauxic shift cultures (for high resolution see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.s007" target="_blank">S7 Fig</a>). An experiment with a higher coverage is shown. Underlying numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.s012" target="_blank">S1 Data</a>. (E) Northern blot analysis of RNA isolated form wild-type yeast or strains deleted for different exosome/TRAMP complex factors or Xrn1. All strains were grown until their growth stopped after the diauxic shift. The <i>xrn1Δ</i> strain stopped growing at the indicated lower OD₆₀₀ than the other strains.