Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

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1 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 prerrna) 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 nop53 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

2 bar, 5 µm. (d) Whole cell lysates derived from nop53 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.

3 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%.

4 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

5 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.

6 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 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.

7 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

8 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.

9 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.

10 Supplementary Table 1. Plasmids used in this Study Name Relevant information Source prs316-nop53 CEN, URA3, PNOP53, TNOP53 Ref. 9 YCplac111-TAP-Flag-NOP53 CEN, LEU2, PNOP53, TADH1, N-terminal TAP-Flag tag Ref. 9 YCplac111-TAP-Flag-nop53 5 Ala CEN, LEU2, PNOP53, TADH1, N-terminal TAP-Flag tag Ref. 9 YCplac111-NOP53 CEN, LEU2, PNOP53, TADH1 Ref. 9 YCplac111-nop53 D64R CEN, LEU2, PNOP53, TADH1 Ref. 9 YCplac111-nop53 5 Ala CEN, LEU2, PNOP53, TADH1 Ref. 9 YCplac111-Flag-nop53 D64R CEN, LEU2, PNOP53, TADH1, N-terminal Flag tag YCplac111-Flag-nop53 5 Ala CEN, LEU2, PNOP53, TADH1, N-terminal Flag tag YCplac111-GFP-NOP53 CEN, LEU2, PNOP53, TADH1, N-terminal GFP tag YCplac111-GFP-nop53 D64R CEN, LEU2, PNOP53, TADH1, N-terminal GFP tag YCplac111-GFP-nop53 5 Ala CEN, LEU2, PNOP53, TADH1, N-terminal GFP tag YCplac111-PGAL1-10-NOP53 CEN, LEU2, PGAL1-10, TADH1 Ref. 9 YCplac111-PGAL1-10-nop53 5 Ala CEN, LEU2, PGAL1-10, TADH1 Ref. 9

11 Supplementary Table 2. Yeast Strains used in this Study Name Relevant genotype Source W303 wild type Ref. 80 LAS1-HA-Aid LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- nop53δ shuffle nop53::natnt2, prs316-nop53 Ref. 9 nop53δ shuffle nop53::his3mx6, prs316-nop53 Ref. 9 nop53δ shuffle nop53::hphnt1, prs316-nop53 NSA3-GFP LAS1-HA-Aid NOP7-GFP LAS1-HA-Aid NOP53-GFP LAS1-HA-Aid NUG2-GFP LAS1-HA-Aid NSA3-GFP::natNT2, LAS1-HA-Aid::His3MX6, P ADH11 -OsTIR1- NOP7-GFP::natNT2, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- NOP53-GFP::natNT2, LAS1-HA-Aid::His3MX6, P ADH11 -OsTIR1- NUG2-GFP::natNT2, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- nop53δ shuffle NSA3-GFP nop53::his3mx6, prs316-nop53, NSA3-GFP::natNT2 nop53δ shuffle NOP7-GFP nop53::his3mx6, prs316-nop53, NOP7-GFP::natNT2 nop53δ shuffle NUG2-GFP nop53::his3mx6, prs316-nop53, NUG2-GFP::natNT2 nop53δ shuffle RPF2-GFP nop53::his3mx6, prs316-nop53, RPF2-GFP::natNT2 REI1-TAP LAS1-HA-Aid REI1-TAP::natNT2, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- nop53δ shuffle REI1-FTpA nop53::his3mx6, prs316-nop53, REI1-FTpA::natNT2 RQC2-TAP NSA3-Flag LAS1- HA-Aid RPL24A-FTpA LAS1-HA-Aid RQC2-TAP:: natnt2, NSA3-Flag::klURA3, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- RPL24A-TAP::natNT2, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- nop53δ shuffle RPL24A-FTpA nop53::his3mx6, prs316-nop53, RPL24A-FTpA::natNT2 RQC2-FTpA LAS1-HA-Aid RQC2-FTpA::natNT2, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- nop53δ shuffle RQC2-TAP nop53::natnt2, prs316-nop53, RQC2-TAP:: His3MX6 RPL24A-FTpA RPL24A-FTpA::His3MX6 TAP-Flag-Nop53 LAS1-HA-Aid P NOP53 -TAP-Flag-NOP53::natNT2, LAS1-HA-Aid::His3MX6, P ADH11 - OsTIR1- nop53δ shuffle rqc2δ rqc2::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle ltn1δ ltn1::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle rqc1δ rqc1::natnt2, nop53::hphnt1, prs316-nop53

12 nop53δ shuffle xrn1δ xrn1::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle dom34δ dom34::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle hel2δ hel2::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle asc1δ asc1::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle ski2δ ski2::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle ski3δ ski3::natnt2, nop53::hphnt1, prs316-nop53 nop53δ shuffle ski7δ ski7::natnt2, nop53::hphnt1, prs316-nop53 rqc2δ rqc2::natnt2 ltn1δ ltn1::natnt2 rqc1δ rqc1::natnt2 xrn1δ xrn1::natnt2 dom34δ dom34::natnt2 hel2δ hel2::natnt2 asc1δ asc1::natnt2 ski2δ ski2::natnt2 ski3δ ski3::natnt2 ski7δ ski7::natnt2 ASC1-FTpA LAS1-HA-Aid SKI2-FTpA LAS1-HA-Aid ASC1-FTpA::natNT2, LAS1-HA-Aid::His3MX6, P ADH11 -OsTIR1- SKI2-FTpA::natNT2, LAS1-HA-Aid::His3MX6, P ADH1 -OsTIR1- NOP7-GFP NOP7-GFP::His3MX6 NSA3-GFP NSA3-GFP::His3MX6 NUG2-GFP NUG2-GFP::His3MX6 NOP7-GFP ski2δ ski2::natnt2, NOP7-GFP::His3MX6 NSA3-GFP ski2δ ski2::natnt2, NSA3-GFP::His3MX6 NUG2-GFP ski2δ ski2::natnt2, NUG2-GFP::His3MX6 RPL24A-FTpA ski2δ ski2::natnt2, RPL24A-FTpA::His3MX6 NSA3-Flag NSA3-Flag::His3MX6 NSA3-Flag ski2δ ski2::natnt2, NSA3-Flag::His3MX6