Diazaborine treatment results in rapid relocalization of shuttling proteins from the nucle(ol)us into the cytoplasm. (A) Scheme demonstrating the effect of diazaborine on the pre-60S maturation pathway after short term and long term treatment. After short term treatment, cytoplasmic pre-60S particles, which are beyond the diazaborine sensitive maturation step at drug application, are able to complete maturation, while particles in maturation stages prior to the diazaborine sensitive step become entrapped, leading to two different cytoplasmic populations of pre-60S particles. This is symbolized by a gap in the cytoplasmic bars. Proteins associating with the pre-60S particle at different maturation stages which were used as bait proteins in this study (Noc2, Nsa1, Rix1) are symbolized by green bars. The individual pre-rRNAs are indicated in blue. Asterisks highlight blocked particles and particles in transition after short term treatment for the Noc2 and Nog1 bait proteins. 25S* and 5.8S* denotes the mature 25S and 5.8S rRNA including their immediate precursors, the 25.5 and 6S pre-rRNA, respectively. (B) Yeast strains expressing GFP fusion with the shuttling protein Nog1 or the nuclear resident protein Rix1 were treated with diazaborine for 30 min and inspected by fluorescence microscopy. For compartment tracking the strains also expressed a mCherry fusion with the nucleoplasmic protein histone H1 (Hho1). (C) Nog1-GFP was expressed in strains harboring mCherry fusions with the nucleolar protein Nop58 or the nuclear membrane protein Nic96 and inspected by laser scanning microscopy after different treatment periods with diazaborine. Only a section of a single scanning plane is shown. In addition, GFP-fusions with the late joining shuttling protein Bud20-GFP were expressed in the Nic96-mCherry background and treated with the inhibitor. A GFP-fusion with the nuclear resident protein Rix1 served as a control. (D) The distribution of the GFP fusions within the cell after diazaborine treatment was estimated based on co-localization of Nop58-mCherry (nucleolus) or on localization within the Nic96-mCherry ring structure (nucleus) throughout the whole stack of scans using the program Fiji (). Nog1 nucleus (cyh): nuclear signal of Nog1-GFP after additional treatment with cycloheximide. Data points representing the average of at least three biological replicates and standard deviations are shown.

Compositional changes of Nog1-TAP containing pre-60S particles during their transition through the maturation cascade. Pre-60S particles were purified from the Nog1-TAP strain after different time periods of treatment with diazaborine. After purification, aliquots were used for protein and RNA extraction as well as iTRAQ analyses. (A) Northern blot analyses with probes hybridizing to the indicated pre-rRNAs. The blots with 27SA₂ pre-rRNA are shown in normal and long exposure time to highlight changes occurring over time. 27SA₂ was detected with the A2A3 probe. Total 27S pre-rRNA and 7S pre-rRNA are from the same exposure and were detected with the E-C2 probe. The other probes were specific for the mature 5.8S, 25S or 18S rRNAs (see for details). (B) Proteins present in the pre-60S particles were analyzed by SDS-PAGE. Proteins showing altered levels in the different preparations were identified by ESI-MS. (C) Western blotting: the Nog1-TAP preparations were analyzed by western blotting with antibodies directed to selected maturation and export factors from various steps of the pathway. (D) Proteins present in the samples were subjected to semi-quantitative iTRAQ analyses. The iTRAQ ratios for each maturation factor were normalized to the iTRAQ ratio of the bait protein and are displayed as a heat map. Mean iTRAQ ratios for small (rpS) and large (rpL) ribosomal proteins as well as translation factors (trans), snoRNPs and housekeeping proteins (housekp) are indicated. Depletion or enrichment of factors is expressed as a color gradient of blue or yellow, respectively (see color code). Numbers in brackets behind protein names indicate the average number of peptides by which the respective protein was identified. Data represent one representative biological replicate. Samples from individual treatment periods were labeled in two series with the 15 min samples present in both sets.

Impact of blocking shuttling protein recycling on pre-rRNA composition of nucleolar and nucleoplasmic pre-60S particles. RNA was extracted from an aliquot of the purifications shown in this figure, separated by agarose gel electrophoresis and analyzed by northern blotting using radiolabeled oligonucleotides directed to the indicated precursor rRNAs. For comparison, northern blot analysis of total RNA from the crude extract (CE) is also shown.

Impact of blocked shuttling protein recycling on composition of nucleolar pre-60S particles. Particles were purified using IgG coated magnetic beads with TAP-tagged nucleolar (Noc1, Nsa1), nucleoplasmic (Rix1) and shuttling proteins (Arx1) as bait proteins and analyzed by SDS-PAGE, western blotting and iTRAQ. (A) SDS-PAGE of TAP purifications after treatment of the yeast strains for 0–60 min. Protein bands differing between treated and untreated samples in the SDS-PAGE were identified by ESI-MS. Asterisks denote bait proteins. (B) Western blot analyses with selected maturation and export factors are shown. (C) Proteomic analysis of purified pre-60S particles by semi-quantitative mass spectrometry using iTRAQ reagents. Mean iTRAQ ratios for small (rpS) and large (rpL) ribosomal proteins as well as housekeeping proteins (housekp) are indicated. The iTRAQ ratios for each maturation factor after 5, 30 and 60 min of drug treatment were normalized to the iTRAQ ratio of the respective bait proteins and displayed as a heat map (see color code). Numbers in brackets behind protein names indicate the average number of peptides by which the respective protein was identified; numbers in squared brackets display the iTRAQ ratios for selected proteins. Samples were derived from the same purification and biological replicate as in Figure and panels (A and B).

The extent of depletion of individual pre-60S maturation factors after diazaborine treatment is linked to their position on the pre-60S particle. Projection of the results of the iTRAQ analyses on the structure of a late nuclear pre-60S particle. The structure of the recently described Nog2 containing particle () was used to color the maturation factors according to the extent of their depletion after 15 min of diazaborine treatment. A similar color scheme as for the iTRAQ analysis was used with factors depleted in different shades of blue and factors enriched in yellow. Factors behaving similar as the bait protein Nog1 are colored in dark gray. Data are based on iTRAQ values representing the mean of two independent biological replicates. Only factors with standard deviations of <30% were considered (). Factors not addressed (ribosomal proteins) or not detected in at least three iTRAQ analyses are shown in light gray.