The assembly of large multimeric complexes in the crowded cytoplasm is challenging. Here we reveal a mechanism that ensures accurate production of the yeast proteasome, involving ribosome pausing and co-translational assembly of Rpt1 and Rpt2. Interaction of nascent Rpt1 and Rpt2 then lifts ribosome pausing. We show that the N-terminal disordered domain of Rpt1 is required to ensure efficient ribosome pausing and association of nascent Rpt1 protein complexes into heavy particles, wherein the nascent protein complexes escape ribosome quality control. Immunofluorescence and in situ hybridization studies indicate that Rpt1- and Rpt2-encoding messenger RNAs co-localize in these particles that contain, and are dependent on, Not1, the scaffold of the Ccr4–Not complex. We refer to these particles as Not1-containing assemblysomes, as they are smaller than and distinct from other RNA granules such as stress granules and GW- or P-bodies. Synthesis of Rpt1 with ribosome pausing and Not1-containing assemblysome induction is conserved from yeast to human cells.
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The working realization of the algorithm for ribosome peak detection is available at https://github.com/fedxa/RiboPeaks. The quantification data shown in Figs. 2e and 5e, and Supplementary Figs. 2a and 5b are available in Supplementary Table 1. Uncropped images and replicate experiments for Figs. 2–6 and Supplementary Figs. 2, 3, 4, 5c, and 8c are available in Supplementary Dataset 1. Any other data are available on request. The ribosome-profiling data are available in the SRA with the accession code PRJNA512900 at the following link: https://www.ncbi.nlm.nih.gov/sra/PRJNA512900.
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We would like to dedicate this manuscript to our co-author Cohue Pena, who sadly passed away just 10 days before acceptance of the manuscript. We thank L. Maillard and S. Zahoran for expert technical assistance. We thank R. Green and her laboratory, in particular A. Radhakrishnan and K. Wehner, for their help in acquiring expertise in ribosome profiling and data analysis. We thank M. Pool, University of Manchester, for antibodies; M. Escobar, Köln University, and D. Finley, Harvard Medical School, for strains; M.E. Gleave, Vancouver Prostate Center, for cell lines; R. Ioris and R. Coppari, University of Geneva, for cell lines and help with cell cultures; and D. Martinvalet for a critical reading of this manuscript. This work was supported by grants from the L’Oreal, Ernst and Lucie Schmidheiny, and Swiss Life Foundation awarded to O.O.P. and by grant nos. 31003a_135794 and 31003A_172999 from the Swiss National Science, and grant no. 15A043 from the Novartis Foundation, awarded to M.A.C. V.G.P. is supported by grants from the Swiss National Science Foundation (no. 31003A_166571), NCCR RNA & Disease, Novartis Foundation, Olga Mayenfisch Stiftung and a Starting Grant Award from the European Research Council (no. EURIBIO260676).
The authors declare no competing interests.
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Integrated supplementary information
We obtained between 1 and 2 million mapped reads in the duplicated samples. a. CDS coverage was compared between replicates after normalizing for library size, showing a high correlation. b. Sample quality was assessed according to footprint length with an average fragment size of 28 nucleotides as expected. c. Footprints displayed the expected 3-nucleotide periodicity. d. Peaks of ribosome pauses were evaluated genome wide using a custom algorithm (see Online Methods). We plotted the distribution of the peaks according to their significance (-log10 p-value). Ribosome pausing on RPT1 with codon D241 at the ribosome P-site and on RPT2 with codon D165 in the ribosome P-site of the ribosome, indicated on the plot, are amongst the 33 most relevant ribosome pause sites genome-wide. 4 other peaks were significant for RPT1 and are indicated.
Supplementary Figure 2 Analysis of various RNCs reveals differences in particle formation, RNC stability, and ribosome stalling.
a. Extracts from copper induced cells expressing Flag-Rrp41–120 K12 or Flag-Rps31–120 K12 or Rpt11–120 K12 at the indicated time points after being treated with CHX were analyzed by immunoblotting with antibodies to Flag and Rpl35. One representative experiment is shown. Quantification of Flag to Rpl35 signal using biological duplicates (n = 2) is shown on the right. Error bars represent the range of the 2 values. b. Extracts from copper induced cells expressing the Rpt2-RNC or the ΔN-Rpt2-RNC as indicated and treated with CHX to preserve polysomes were analyzed by sucrose gradient sedimentation. The indicated fractions were analyzed by immunoblotting with antibodies to Flag and Rpl35 (left panels). The Ponceau staining is shown below the blots. The samples were probed with antibodies to V5 and on the right are shown comparisons of the signal with antibodies to Flag and V5 using comparable exposures. c. Extracts from copper induced cells expressing HA-Rpt2 1–166 were analyzed by sucrose gradient sedimentation. Free (F, lanes 1,2), Monosome (M, lanes 3,4) light polysome (P1, lanes 5,6) and heavy polysome (P2, lanes 7,8) fractions were analyzed by immunoblotting with antibodies to HA and Rpl35. d. Extracts from copper induced cells expressing the Rpt1-RNC or Rpt2-RNC as indicated were analyzed by immunoblotting with antibodies to Flag or V5. e. Total extracts from cells expressing the Rpt2-RNC and ΔN-Rpt2-RNC after copper induction were prepared by post-alkaline lysis and equal cell-equivalent amounts (lanes 1 and 4, 6 μl; lanes 2 and 5, 9 μl and lanes 3 and 6, 12 μl) were analyzed by immunoblotting with antibodies to Flag and Rpl35 as indicated. The Ponceau stain of the gel is shown below. f. Extracts from copper-induced cells expressing the HA-ΔN-Rpt1 and HA-Rpt1 were prepared by post-alkaline lysis. Three different cell-equivalent amounts were analyzed by immunoblotting with antibodies to HA and Rpl35.
Cells expressing Rpn11-ProtA were transformed with plasmids expressing the Rpt1- or Rpt2-RNC and HA-Rpt2 or HA-Rpt1, or with empty vectors (-) with the same marker genes URA3 or LEU2 as indicated. After 10 min copper induction extracts were prepared and incubated with IgG-sepharose beads. Rpn11 was eluted by treatment of the beads with TEV protease. The input (TE) and TEV eluate (Eluate) were analyzed by immunoblotting. One blot was probed with successively anti-Rpt2 (a), followed by anti-HA (b) and finally anti-Flag (c). Hence the signal in panel c cumulates the signals in panels a and b. One blot was probed only with anti-Rpt1 (d). The positions of the detected RNCs (Flag-tagged, see Fig. 1d), HA-tagged proteins or tagged Rpn11 are indicated on the right, while MW markers are indicated on the left. In panel a, the polyclonal antibodies to Rpt2 will recognize endogenous Rpt2, HA-Rpt2 and the Rpt2-RNC. In this panel the signal for HA-Rpt2 in the TE is overshadowed by a signal from Rpn11-ProtA. In panel b, we still see the signals from panel a, but we additionally see the signals from the HA antibodies. This reveals the position of HA-Rpt2 in the first TE lane not clearly seen in panel a and the low levels of HA-Rpt2 in the first Eluate lane that could not be seen with Rpt2 antibodies in panel a. HA-Rpt1 migrates nearly at the same level as Rpn11-ProtA and hence cannot be easily distinguished in the second TE lane, but a very small amount of HA-Rpt1 can be guessed in the second Eluate lane. Finally in panel c, we still see the signals from panels a and b, but we additionally see low amounts of Rpt2-RNC (Flag-tagged, see Fig. 1d) in the second Eluate lane that could not be seen with antibodies to Rpt2 (in the second Eluate lane of panel a) and of the Rpt1-RNC (Flag-tagged, see Fig. 1d) in the first Eluate lane that could not be seen with antibodies to Rpt1 (in the first Eluate lane of panel d). Although in these experiments the signals of HA-Rpt1 and HA-Rpt2 could not be easily be compared to the signals of the endogenous subunits because of interference from the signal arising from Rpn11-ProtA, this could be assessed in cells that do not express tagged Rpn11 (blots available in the Supplementary Dataset 1). HA-Rpt2 was expressed at levels similar to endogenous Rpt2, whereas HA-Rpt1 was less expressed than the endogenous Rpt1.
a. Extracts from cells induced by copper and expressing Flag-Rpt1 and HA-Rpt2 or Flag-Rpt2 and HA-Rpt2 were incubated with antibodies to Flag (IP-Flag), HA (IP-HA) or Myc (IP-Myc) as a negative control. The total extract and immunoprecipitates were analyzed by immunoblotting with antibodies to HA, Flag and Not1. b. The indicated strains were grown exponentially and serially diluted. 4 μl of each dilution were spotted on rich medium (YPD), or on rich medium containing hygromycin B (HYG, 0.1 mg/ml) or azetidine-2 carboxylic acid (AZC, 0.5 mg/ml) as indicated. The cells were left to grow for 3 days at 30 °C. c. Extracts from cells induced by copper and expressing the Rpt1-RNC and HA-Rpt275–166 treated with CHX to preserve polysomes were analyzed by sucrose gradient sedimentation. The indicated fractions were analyzed by immunoblotting with antibodies to HA and Flag. d. Extracts from cells induced by copper and expressing the Rpt1-RNC or ProtA-Rpt1-DP and HA-Rpt275–166 were incubated with anti-Flag IgG beads overnight. The flow through was collected (FT) and then after washing Flag peptide was added to elute the RNC-associated proteins (IP Flag). Total extract (TE), IP Flag and FT were analyzed by immunoblotting with antibodies to Flag and HA. Biological duplicates were analyzed.
a. The patterns of ribosome footprints aligned on the P-site of the ribosome for PSMC2 encoding human Rpt1 was extracted from published data29. The P-site codon, 135, where footprints accumulate, is indicated. The conserved N-terminal helix of Rpt1 that interacts with an N-terminal helix of Rpt2 in the mature proteasome (aa 45–71) is depicted in red. b. Extracts from LNCaP cells untreated or treated with arsenite were separated on a sucrose gradient. RNA from the total extract or from the indicated fractions was prepared and analyzed for the levels of PMSC2 or CDK16. Normalization was to the levels of EIF4E2 mRNA. The experiment was performed in biological duplicates (n = 2) and the data for quantification is available in Supplementary Table 1. Error bars represent the range of the 2 values. c. Extracts from A549 lung cancer cells treated or not with arsenite were incubated with EDTA for 1 h and fractionated on sucrose gradients. The total extract (TE) and indicated fractions from the polysome profiles shown on the right were analyzed by immunoblotting with anti-CNOT1 antibodies. This experiment was performed only once.
a. NCA are formed in response to different stress conditions. LNCaP cells were treated with oxidative stress inducer arsenite (100 μM), endoplasmic reticulum (ER) stress inducer thapsigargin (1 μM), proteasome inhibitor MG-132 (10 μM) and UV radiation for 1 h. Cells were fixed and subjected to IF using the indicated antibodies. b. CHX treatment didn’t affect NCA. LNCaP cells were treated with 100 μM arsenite and 10 μM cycloheximide (CHX) for 1 h. Cells were fixed and subjected to IF using the indicated antibodies. c. YB-1 is used as a stress granule marker to differentiate CNOT1 containing particles. Untreated or arsenite (100 μM for 1 h) treated cells were processed for IF as described above using the indicated antibodies. d. NCA are distinct from P-bodies and GW bodies. LNCaP cells treated with 100 μM arsenite for 1 h were fixed and subjected to IF using anti-DCP1A and anti-DDX6 antibodies (P body markers) and anti-GW182 antibodies (GW bodies). The slides were co-stained with anti-CNOT1 antibodies. Note that NCA stay distinct from P bodies and GW bodies. e. NCA are present in different cell lines. 22Rv1 (three upper panels) and V16D (bottom three panels) prostate cancer cells were treated with 100 μM arsenite for 1 h and subjected to IF using the indicated antibodies. Note that NCA are detected in both cell lines. f. In situ hybridization using mismatch probes targeting Rpt1 and Rpt2 in LNCaP cells. g. LNCaP cells treated with 100 μM arsenite for 1 h were fixed and subjected to in situ hybridization using 56-FAM-labeled oligos targeting Rpt1- or Rpt2-encoding mRNAs. Following hybridization, cells were immunostained with anti-CNOT1 antibodies. h. LNCaP cells treated with 100 μM arsenite or 100 μM arsenite and CHX for 1 h were subjected to in situ hybridization using differently labeled oligos targeting Rpt1- or Rpt2-encoding mRNAs to look for co-localization. i. LNCaP cells transfected with siControl or siCNOT1 siRNAs were treated with 100 μM arsenite for 1 h and subjected to IF using the indicated antibodies. j. LNCaP cells transfected with siCNOT1 siRNAs were treated with 100 μM arsenite for 1 h. The cells were fixed and subjected to in situ hybridization using differently labeled oligos targeting Rpt1- or Rpt2- encoding mRNAs to look for co-localization. Note that Rpt1 and Rpt2 co-localization is generally lost in the absence of CNOT1. Scale bar 10 μm. Each experiment was conducted independently at least 3 times. 10–15 images were captured for each condition.
The red box indicates the position of the codons orthologous/paralogous to the DP pause site at codon 165 of Rpt2. The green box indicates the position of the codons orthologous/paralogous to the DP pause site at codon 241 of Rpt1. The blue box indicates the codons orthologous/paralogous to the D at position 135 of Rpt1.
a. Arsenite (100 μM for 1 h) treated LNCaP cells were processed for IF as described above using antibodies to CNOT6 or CNOT1 as indicated. b. LNCaP cells untreated (UT) or treated with 100 μM arsenite (ARS) for 1 h were fixed and subjected to in situ hybridization using 56-FAM-labeled oligos targeting Rpt1- or Rpt2-encoding mRNAs. Following hybridization, cells were immunostained with anti-CNOT6 antibodies. Each experiment was conducted independently at least 3 times. 10–15 images were captured for each condition. c. Extracts from not4Δ or not5Δ cells induced by copper and expressing the Rpt1-RNC at the indicated times after being treated with CHX were analyzed by immunoblotting with antibodies to Flag and Rpl35. These results were obtained independently more than 3 times.
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Panasenko, O.O., Somasekharan, S.P., Villanyi, Z. et al. Co-translational assembly of proteasome subunits in NOT1-containing assemblysomes. Nat Struct Mol Biol 26, 110–120 (2019). https://doi.org/10.1038/s41594-018-0179-5
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