Abstract
Assembly of fully functional ribosomes is a prerequisite for failsafe translation. This explains why maturing preribosomal subunits have to pass through an array of quality-control checkpoints, including nuclear export, to ensure that only properly assembled ribosomes engage in translation. Despite these safeguards, we found that nuclear pre-60S particles unable to remove a transient structure composed of ITS2 pre-rRNA and associated assembly factors, termed the 'foot', escape to the cytoplasm, where they can join with mature 40S subunits to catalyze protein synthesis. However, cells harboring these abnormal ribosomes show translation defects indicated by the formation of 80S ribosomes poised with pre-60S subunits carrying tRNAs in trapped hybrid states. To overcome this translational stress, the cytoplasmic surveillance machineries RQC and Ski-exosome target these malfunctioning ribosomes. Thus, pre-60S subunits that escape nuclear quality control can enter translation, but are caught by cytoplasmic surveillance mechanisms.
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Acknowledgements
We would like to thank O. Berninghausen and C. Ungewickell for cryo-EM data collection and vitrifying samples, as well as P. Ihrig and J. Reichert (BZH) for performing all MS analyses. We are grateful to A.W. Johnson (University of Texas at Austin), B. Stillman (Cold Spring Harbor Laboratory), S. Ottonello (Università di Parma), J.R. Warner (Albert Einstein College of Medicine), J.P. Ballesta (Centro de Biologia Molecular Severo Ochoa), M. Fromont-Racine (Institut Pasteur), M. Seedorf (ZMBH University of Heidelberg), V. Panse (University of Zurich), B.L. Trumpower (Dartmouth Medical School) and E. Tosta for their gift of antibodies. We also thank C. Joazeiro (ZMBH University of Heidelberg) for critically reading the manuscript. E.H. is a recipient of grants from the Deutsche Forschungsgemeinschaft DFG (HU363/10-5, HU363/12-1).
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M.T., A.S., C.B.-G., R.B. and E.H. designed the study and analyzed the data. A.S. and M.T. performed all experiments except cryo-EM, which was done by C.B.-G. and R.B. E.T. performed the northern blot presented in Supplementary Figure 5c. D.F. performed the negative-stain EM. A.S., M.T., C.B.-G., E.T., R.B. and E.H. interpreted the results and wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Supplementary Figure 1 Impaired ITS2 processing results in mislocalization of foot-factors to the cytoplasm.
(a) Schematic representation of ITS2 processing. The endonuclease Las1 initiates ITS2 processing by cleaving at site C2 (of the pre-rRNA) that leads to the separation of the 7S and the 26S rRNA pathways. The 7S pathway proceeds by Nop53 mediated recruitment of Mtr4 and the exosome in the nucleus, which finally leads to mature 5.8S (in the cytoplasm) via different intermediates (indicated in the scheme). (b) Western blot analysis of whole cell lysates from the Las1-Aid strain (upper panel) and the Las1-Aid Nsa3-Flag Rqc2-TAP strain (lower panel), under conditions of Las1 expression (- Auxin) and Las1 depletion (+ Auxin). Samples were probed with the indicated antibodies. The results show that the levels of foot factors remain unperturbed upon Las1 depletion. The arrow points to the western blot signal of the HA tagged Las1-Aid and the asterisk indicates a background signal from the HA antibody (lower panel). (c) The subcellular localization of the GFP-tagged Nop7, Nsa3, Nug2 and Rpf2 from nop53Δ yeast cells under the expression of plasmid borne NOP53 wild-type, nop53 5×Ala and nop53 D64R mutant alleles. Cells were analyzed by fluorescence and DIC microscopy. Scale bar, 5 μm. (d) Whole cell lysates derived from nop53Δ yeast cells harboring the N-terminal TAP-Flag tagged NOP53 wild-type or the nop53 5×Ala allele, where analyzed by sucrose gradient centrifugation. The 40S, 60S, 80S and polysome fractions are indicated and halfmers are marked by arrowheads. The sucrose gradient fractions were analyzed by western blot analysis and probed with anti-ProtA (Nop53) and anti-L3 antibodies.
Supplementary Figure 2 Negative-stain EM of the Rqc2-TAP Nsa3-Flag particle under Las1 depletion conditions in comparison to mature 60S particles.
(a) Coomassie stained SDS-PAGE of the split purified Rqc2-TAP Nsa3-Flag particle obtained after depletion of Las1-Aid (+ Auxin, 2 h), which was used for subsequent negative stain EM analysis. (b) Selected 2D class averages of the Rqc2-TAP Nsa3-Flag particle purified from Las1 depleted cells. The class average shown in Fig. 3d is marked with a red box. Arrowheads point to a diffuse density, which might correspond to Ltn1. (c) Selected 2D class averages of an L24A-FTpA particle, which is a mixture of mature 60S subunits and 80S ribosomes. The population of the mature 60S subunit particle (marked with a red box) in the total dataset is ~3%.
Supplementary Figure 3 Cryo-EM sorting scheme of the TAP-Flag-Nop53 particle obtained after Las1 depletion.
The particles obtained from the TAP-Flag-Nop53 purification after Las1 depletion were classified using iterative multi-reference projection alignment. The data was sorted into 5 different classes, all of them displaying density for the foot structure. Class 2, 3 and 5 showed a strong density for the 60S subunit, but only very noisy density in the 40S region. Classes 1 and 4 represented 80S ribosomes in the absence or presence of tRNAs, respectively. These two classes were taken together for a new round of classification that gave the final classes containing empty 80S and 80S with tRNAs. Then, the final classes were refined using only the particles with higher cross correlation. The final structure is highlighted in green. Other classification rounds were performed with different approaches in order to prove that particles were fully classified (see Online Methods). In the final classes the density for the 40S subunit is displayed in yellow, the A/P tRNA in green, the foot structure in orange and eIF6 in red.
Supplementary Figure 4 Resolution of the foot containing 80S particle with comparison to peptidyl-tRNA-60S subunits bound to Rqc2 and Ltn1.
(a) Overall resolution measured at the 0.143 cut-off of the FSC from two independent datasets. The final subpopulation was refined to a resolution of 7.3 Å. (b) Nop53 Las1-depleted 80S map colored according to its local resolution. (c) Comparison between the cryo-EM structure of peptidyl-tRNA-60S ribosomes bound to Rqc2 and Ltn1 (Shen, P.S. et al., Science. 347, 75-8, 2015) (EMDB-6170; overall structure in light blue, Ltn1 and Rqc2 are respectively highlighted in tan and purple) and the reconstruction of the foot-containing 80S particle (gray, foot structure shown in orange). For easier visualization the 40S subunit of the latter reconstruction was omitted.
Supplementary Figure 5 The nop53 5×Ala mutant is genetically and functionally linked to cytoplasmic surveillance factors.
(a) Growth analysis comparing a wild-type strain and the indicated deletions strains under galactose overexpression of plasmid-based NOP53 wild-type or nop53 5×Ala mutant alleles (under control of the GAL1-10 promoter). An empty vector served as control. Cells were spotted in 10-fold serial dilutions on SDC-Leu (glucose) and SGC-Leu (galactose) medium and cell growth at 30°C was monitored after 2 and 3 days, respectively. (b) Polysome gradient analysis of whole cell lysates derived from a Nsa3-Flag and Nsa3-Flag ski2Δ strain after overexpression (for 8 h) of the dominant negative GAL::nop53 5×Ala mutant. The fractions containing 40S, 60S, 80S and polysomes are indicated. The sucrose gradient fractions were analyzed by western blot analysis and probed with the indicated antibodies. (c) Analysis of pre-rRNA and rRNAs extracted from the indicated yeast strains. Galactose induction was performed for 8h, following which RNAs were extracted and analyzed by Northern blotting. Oligonucleotide probes used for Northern analysis: precursors to 5.8S rRNA (top panel) were detected with the 020 oligo probe (TGAGAAGGAAATGACGCT) and the 5S rRNA was detected with the 041 oligo probe (CTACTCGGTCAGGCTC). Values shown indicate the relative abundance of 7S pre-rRNA compared to the wild-type lane (1), when normalized to 5S rRNA levels. Asterisk marks the abnormal processing intermediate between 7S and 5.8S+30.
Supplementary Figure 6 Analysis of the foot-containing 80S structure.
(a) Magnification of the foot structure (PDB ID: 3JCT) fitted into the density of the 80S ribosome presented in this study. Factors belonging to the foot structure as well as ITS2 are highlighted in different colors and labeled accordingly. The structure of the 60S subunit (PDB ID: 5TGM) is fitted in order to display the main interactions between the foot structure and the 60S subunit. R-proteins interacting with the foot L8 (eL8), L25 (uL23) and L27 (eL27) are highlighted in green, yellow and red respectively (Wu, S. et al., Nature. 534, 133-7, 2016). 25S and 5.8S rRNAs are shown in gray and the rest of the r-proteins in beige. (b) The fit of the foot containing 80S ribosome into the density of actively translating polysomes (EMDB-2790) demonstrates that the foot (orange) would clash with the small subunit of the next ribosome (light blue). (c) The transition between the two conformations of ES27 (displayed in green and blue, PDB ID: 3IZF and 3IZD) may be obstructed by the presence of the foot.
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Sarkar, A., Thoms, M., Barrio-Garcia, C. et al. Preribosomes escaping from the nucleus are caught during translation by cytoplasmic quality control. Nat Struct Mol Biol 24, 1107–1115 (2017). https://doi.org/10.1038/nsmb.3495
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DOI: https://doi.org/10.1038/nsmb.3495