Analyses of the RNA and protein content of pre-60S particles purified via Nog1-TAP after <i>in</i><i>vivo</i> depletion of selected ribosomal proteins.

<p>The indicated yeast strains expressing a chromosomally-encoded TAP-tagged version of the LSU biogenesis factor Nog1 together with either RPL25, RPL2, RPL43, RPL21 or no LSU r-protein gene under control of the galactose-inducible GAL1/10 promoter were cultivated for four hours in glucose-containing medium to shut down expression of the respective LSU r-protein gene. Nog1-TAP and associated pre-ribosomal particles were then affinity purified from corresponding cellular extracts as described in Materials and Methods. The (pre-) rRNA content of total cellular extracts (Input lanes 1-5) or of parts of the affinity purified fractions (IP lanes 6-10) was analyzed by northern blotting and is shown in (<b>A</b>). Detected (pre-) rRNAs are indicated on the left and oligonucleotides used for (pre-) rRNA detection are indicated on the right. Equal signal intensities of the Input and IP fractions correspond to 1% (35S, 27S pre-rRNAs and 25S and 18S rRNAs) or 10% (7S pre-rRNA, 5.8S, 5S rRNAs and glutamyl-tRNA) co-purification efficiencies, respectively. Purification efficiencies of the bait protein Nog1-TAP were monitored by western blotting (see panel designated Nog1-TAP). Equal signal intensities in the western blot analyses indicate 25% purification efficiency of Nog1-TAP. Quantitation of the co-purification efficiencies (in %) of the 7S, 27S, 25S and 5.8S (pre-) rRNAs are shown in (<b>B</b>). Red bars in (<b>B</b>) designate internal background levels of the affinity purification procedure as measured by co-purification efficiencies of 20S pre-rRNA. One part of the affinity purified fractions was further processed for comparative protein analyses by semi-quantitative mass spectrometry using iTRAQ reagents as described in Materials and Methods. In each experiment particles purified from two strains expressing or not one of the r-proteins of interest were compared. The iTRAQ ratio for each LSU r-protein (<b>C</b>) or LSU biogenesis factor (<b>D</b>) which was identified by more than one peptide (except rpL43, marked by a (*)) in more than 70% of all comparisons are displayed as a heat map (see color code). A grey color indicates that the respective protein was not identified in the individual experiment shown in this lane. iTRAQ ratios in (<b>C</b>) were normalized to the median value of all LSU r-protein ratios. iTRAQ ratios in (<b>D</b>) were normalized to the iTRAQ ratio of the bait protein Nog1-TAP. Numbers in brackets behind protein names indicate the average number of peptides by which the respective protein was identified in a single experiment. In (<b>C</b>) the iTRAQ ratios of the LSU r-protein whose expression had been shut down are highlighted by red boxes. Several biological replicates of individual experiments are shown in (<b>C</b>) and (<b>D</b>). (<b>E</b>) shows a northern blot analysis of the amount of 7S pre-rRNA co-purifying with Noc2-TAP from extracts of a wild type strain or of conditional expression mutants of RPL2 or RPL43. Cultivation of the strains, affinity purification and northern blotting procedures were done as described above. Same signal intensities detected in the Input and IP fractions correspond to 2.2% purification efficiency of the 7S pre-rRNA.