The small and large ribosomal subunits depend on each other for stability and accumulation

Cultures were grown in YEP-galactose medium and shifted to YPD glucose medium for the indicated lengths of time before harvest. (A, B) The control strain BY4741 in galactose and glucose medium, respectively. (C, D) P_gal-uL4 at 0 and 8 h, respectively. (E–H) P_gal-uL22 at 0, 2.5, 8, and 16 h, respectively. (I, J) P_gal-Rpf2 at 0 and 16 h, respectively. (K, L) P_gal-Rrs1 at 0 and 16 h, respectively. For C and D, equal volumes of fractions in the ribosome portion of the gradient were analyzed for r-proteins uS4, uL4, uL5, and uL18 by Western blot. (M, N) Quantification of 40S subunits. The area under each ribosomal peek was measured using ImageJ and normalized to the area under all ribosomal peaks. (M) The fraction of ribosomal mass in 40S subunits was calculated as the fraction found in the free 40S peak plus 1/3 of the fraction in 80S and polysomes. (N) The fraction of ribosomal mass in free 40S subunits. Red and green circles refer to repression of uL22 and uL4 synthesis, respectively. Purple and blue triangles refer to repression of Rpf2 and Rrs1 synthesis, respectively.

Cultures of BY4741, P_gal-uL4, P_gal-uL18, and P_gal-eL40 were grown in YEP-galactose and shifted to YPD. (A–D) Aliquots were harvested at 0, 1, 2, 4, and 8 h and constant A²⁸⁰ units from each sample were analyzed by Western blots probed with antisera for uS4, uL4, uL5, and uL18. The intensities of the Western bands were quantified using ImageJ. The yellow curve in (panel B) indicates the calculated dilution curve after repressing uL4 synthesis. (E–H) Data from A–D were normalized to the values for uL4. (I, J) Ratio between 18S and 25S rRNA. Total RNA was purified using RiboPure (I) or hot phenol extraction (J). Equal A²⁶⁰ units of each sample (0.06 corresponding to ∼3 μg) were applied to agarose gels (I). Bands were blotted to a membrane and stained with methylene blue. (J) Gel was stained with ethidium bromide, photographed on a gel imaging system, and quantified with ImageJ. Images of the Western blots are shown in Fig S3. Fig S4 shows images of the ethidium bromide stained gels.

Source data are available for this figure.

Cultures in YEP-galactose medium were shifted to glucose medium for the indicated lengths of time before harvest. (A, B) P_gal-uS4 at 0 and 8 h, respectively. (C) P_gal-uS7 at 8 h (D–I) P_gal-uS4 at 0, 2, 4, 5, 6, and 8 h, respectively. (J, K) P_gal-Rrp7 at 0 and 16 h, respectively. For A–C, equal volumes of fractions in the ribosome portion of the gradient were analyzed for r-proteins uS4, uL4, uL5, and uL18 by Western blot. (L, M) uL23-GFP synthesized during repression of the uS17 gene is incorporated into 80S ribosomes. P_gal-uS17 harboring an estradiol-inducible uL23-GFP gene was grown in synthetic galactose medium and shifted to synthetic glucose medium. 8 h after the shift, uL23-GFP synthesis was induced for 1 h. The indicated fractions were probed by Western analysis for GFP and uL4 proteins.

Cultures of P_gal-uS4, P_gal-uS11, and P_gal-uS10 were grown in YEP-galactose and shifted to YPD. The control experiment with BY4741 is shown in Fig 3A and E. (A–C) Aliquots were harvested at 0, 1, 2, 4, and 8 h and equal A²⁸⁰ units from each sample were analyzed by Western blots probed with antisera for uS4, uL4, uL5, and uL18. The intensities of the Western bands were quantified using ImageJ. The yellow curve in panel A indicates the dilution curve calculated from the growth curve after repressing uS4 synthesis. (D–F) Data from A–C were normalized to the values for uL4. (G, H) Ratio between 18S and 25S rRNA. Total RNA was purified using RiboPure (G) or hot phenol extraction (H). Equal A²⁶⁰ units of each sample (0.06 corresponding to ∼3 μg) were applied to agarose gels. (G) Bands were blotted to a membrane and stained with methylene blue. (H) Gel was stained with ethidium bromide, photographed on a gel imaging system, and quantified with ImageJ. Images of the Western blots are shown in Fig S3. Fig S4 shows images of the ethidium bromide stained gels.

Source data are available for this figure.

(A–C, F) The control strain BY4741 and strains in which the indicated proteins were expressed from the galactose promoter were grown in YEP-galactose (A–C, F) or synthetic complete galactose medium lacking uracil (panels D, E, and G). At time 0, the cultures were shifted to glucose medium for the indicated time. (A–C) Measurement of the sum of transcripts containing ITS1 and ITS2. Total RNA from BY4741, P_gal-uL4, and P_gal-uS4 was purified, and equal A²⁶⁰ units were loaded onto a nylon membrane in a slot format (A). The membrane was probed with ITS1 oligonucleotide O1663 (B) or ITS2 oligonucleotide O1660 (C); see panel H for the position of the sequences to which they hybridize. (D) Analysis of 5′ ends at sites A2 and A3 in rRNA processing intermediates. ³²P end-labeled primer O1345 (panel G) was hybridized to equal amounts of total RNA from P_gal-uL4 (YLL2083) and extended to 5′ ends of processing intermediates generated by cleavage A2 and A3 (see panel H). (E) Run-on analysis of rRNA transcription during repression of uL4 synthesis. Aliquots of cultures of P_gal-uL4 (YLL2083) growing in synthetic medium were harvested at the indicated times after the shift to glucose medium. Equal A²⁶⁰ units from each sample were used for run-on labeling with α-³²ATP. The products were hybridized to slot blots of pDK16 carrying the 35S rRNA transcription unit (Lindahl et al, 1994). (F) Yield of A²⁶⁰ material per OD₆₀₀ units of culture harvested. Equal OD600 units of P_gal-uL22 and BY4741 were harvested at the indicated times. Total RNA was prepared by the hot phenol method and the A²⁶⁰ of the final preparation was measured and normalized to time 0. (G) Pulse-chase analysis of rRNA processing during repression of uL4 synthesis. P_gal-uL4 (YLL2083) was grown in synthetic medium and harvested 0 or 6 h after the shift to glucose medium. The cells were labeled with ³H-uracil for 2 min and then incubated (chased) with a large excess of nonradioactive uracil for the indicated times. RNA was extracted and equal amounts of radioactivity from each sample were then fractionated by agarose gel electrophoresis, transferred to a membrane, and visualized by fluorography. (H) Map of the yeast Pol I rRNA transcription unit. The black boxes immediately below the map show the positions of the oligonucleotide probes used. The arrows labeled D, A2, A3, and C2 indicate cleavage sites relevant for this study. Other processing sites are omitted. The major rRNA processing intermediates (20S and 27S) are indicated below the map. For more details on rRNA processing, see Woolford & Baserga (2013).

Source data are available for this figure.

P_gal-eS31 or P_gal-eL43, each carrying plasmid-borne genes for uS5-GFP or uL23-GFP expressed from their native promoters, were grown in synthetic galactose medium and shifted to synthetic glucose medium for 16 h. The cells were inspected by confocal microscopy. A uniform distribution of the GFP-tagged protein between the nucleus and cytoplasm indicates that the export of ribosomal precursor particles carrying the tagged protein is normal. A buildup of the GFP-tagged in the nucleus indicates that ribosomal precursor particles carrying the tagged protein are not matured to nuclear export competency.

P_gal-uS4 was grown in YEP-galactose medium, and aliquots were harvested at 0 h and 8 h after the shift to glucose medium. (A, B) Equal A²⁶⁰ units of whole cell extracts were fractionated on sucrose gradients and equal portions of the fractions sedimenting as 40S or larger particles were fractionated on an agarose gel, transferred to a membrane, and stained with methylene blue. (C) Equal portions of fractions from identical positions in a set of sucrose gradients from a biological replicate experiment were loaded into alternate slots of an agarose gel, transferred to a membrane, and probed with a mixture of equal radioactivity of end-labeled oligonucleotides O1680 and O1660 that hybridize to ITS1 (distal to the A2 processing site) and ITS2 (Fig 3H). (D) The membrane was then stripped of radioactivity and probed with an oligonucleotide specific to the 5′ end of 25S rRNA. The positions of the A²⁶⁰ peaks in the gradients are indicated below the images of the membrane in (C, D). (E) Lanes marked with black dots in panel (D) were excised electronically from the image in (D) and optimized for viewing of the individual lanes. (F–G) Northern blots of RNA from the 55S and 60S fractions of a sucrose gradient of samples harvested 8 h after addition of glucose and probed with radioactive oligonucleotides hybridizing to the 5′ (panel F) or the 3′ (panel G) ends of 25S rRNA. Broken red lines to the left of the relevant lanes indicate rRNA degradation products.