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Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit

Nature Structural & Molecular Biology volume 23, pages 1074–1082 (2016)Cite this article

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Abstract

Nuclear export of preribosomal subunits is a key step during eukaryotic ribosome formation. To efficiently pass through the FG-repeat meshwork of the nuclear pore complex, the large pre-60S subunit requires several export factors. Here we describe the mechanism of recruitment of the Saccharomyces cerevisiae RNA-export receptor Mex67–Mtr2 to the pre-60S subunit at the proper time. Mex67–Mtr2 binds at the premature ribosomal-stalk region, which later during translation serves as a binding platform for translational GTPases on the mature ribosome. The assembly factor Mrt4, a structural homolog of cytoplasmic-stalk protein P0, masks this site, thus preventing untimely recruitment of Mex67–Mtr2 to nuclear pre-60S particles. Subsequently, Yvh1 triggers Mrt4 release in the nucleus, thereby creating a narrow time window for Mex67–Mtr2 association at this site and facilitating nuclear export of the large subunit. Thus, a spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit.

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Figure 1: Reconstitution of Mex67–Mtr2 binding to pre-60S ribosomal particles.
Figure 2: Mex67 binds specifically at two distinct sites on the isolated pre-60S particles.
Figure 3: Cryo-EM structure of the Yvh1 particle reveals an absence of Mrt4 and Yvh1 binding close to Rpl12.
Figure 4: Mrt4 acts as a placeholder for transport factor Mex67–Mtr2 on the nuclear pre-60S ribosome before nuclear export.
Figure 5: In vivo Yvh1 is functionally linked to the pre-60S nuclear export machinery.
Figure 6: Model of coupling P0 stalk biogenesis with Mex67–Mtr2 export-factor recruitment.

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Acknowledgements

We would like to thank E. Thomson and S. Gnanasundram for assistance in setting up the CRAC protocol, D. Flemming for performing negative-stain EM and J. Baßler for general scientific input and critical reading of the manuscript. We would also like to thank D. Ibberson at the Cell Networks deep-sequencing core facility for performing MiSeq sequencing and the MS facility (P. Ihrig and J. Reichert) at BZH, Heidelberg, for performing all MS analysis. We are grateful to A.W. Johnson (University of Texas), B. Stillman (Cold Spring Harbor Laboratory), C. Dargemont (INSERM), F. Fasiolo (CNRS France), H. Bergler (Karl-Franzens-Universität Graz), J.R. Warner (Albert Einstein College of Medicine), J.P. Ballesta (Centro de Biologia Molecular Severo Ochoa), M. Fromont-Racine (Institut Pasteur), M. Remacha (Centro de Biologia Molecular Severo Ochoa) and V. Panse (ETH Zurich) for their gifts of antibodies. E.H. is supported by grants from the Deutsche Forschungsgemeinschaft (DFG; HU363/10-5, HU363/12-1).

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  1. Anshuk Sarkar and Markus Pech: These authors contributed equally to this work.

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  1. Biochemistry Centre, University of Heidelberg, Heidelberg, Germany

    Anshuk Sarkar, Matthias Thoms & Ed Hurt

  2. Gene Center, University of Munich, Munich, Germany

    Markus Pech & Roland Beckmann

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Contributions

A.S., R.B. and E.H. designed the study and analyzed the data. A.S., performed all experiments except cryo-EM, which was performed by M.P.; R.B. and M.T. generated the strains. A.S., M.P., R.B. and E.H. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Roland Beckmann or Ed Hurt.

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Integrated supplementary information

Supplementary Figure 1 In vitro reconstitution of Mex67–Mtr2 binding to the pre-60S particle.

(a) Binding of recombinant Mex67-Mtr2 to the isolated Arx1 pre-60S particle. SDS-PAGE and Coomassie staining (upper panel) or western blotting (lower panel; a-Mex67 antibody) of increasing amounts of purified Mex67-Mtr2 (lane 1-5; input) and the final Arx1-FTP Flag eluates obtained after incubation without Mex67-Mtr2 (lane 6) or increasing amounts of Mex67-Mtr2 (lanes 7-11). (b) In vitro binding of E. coli expressed and purified Mex67-Mtr2 (lane 1) to the indicated pre-60S particles, affinity-purified via bait proteins Nsa1-FTP, Rsa4-FTP, Yvh1-FTP and Lsg1-FTP in the absence (lane 2-5; Flag eluates) or presence of recombinantly added Mex67-Mtr2 (lane 6-9). Coomassie-stained SDS-polyacrylamide gel (upper panel) and western blot using α-Mex67 antibodies (lower panel). Asterisk (*) marks the position of the respective Coomassie stained bait protein and arrowhead shows the in vitro bound Mex67 band.

Supplementary Figure 2 CRAC analysis of bound rRNA.

(a) In vitro CRAC of recombinant His-tagged Mex67-Mtr2 reconstituted onto the purified Yvh1-FTP particle. Yvh1-FTP was incubated without or with His-tagged Mex67-Mtr2. Rsa4-FTP served as control for unspecific binding by also incubating it with His-tagged Mex67-Mtr2. After binding and washing, 10% of the flag eluates (the rest was used for in vitro UV crosslinking) were analyzed by SDS-PAGE and Coomassie staining. Asterisk (*) marks the position of the respective Coomassie stained bait protein and arrowhead shows the in vitro bound Mex67 band. (b) Autoradiogram of covalent His-Mex67-His-Mtr2::RNA complex after UV-crosslinking (1h exposure). Digested RNA crosslinked to His-tagged Mex67-Mtr2 bait was end-labeled with γ32P-ATP. Red box shows the migration of the protein:RNA complex on the gel, which was later excised and used for RNA extraction, cDNA amplification and sequencing. (c) Same as (b), but the contrast was increased via FIJI image manipulation tool (Schindelin, J. et al., Nat Methods 9, 676-682, 2012). A background Mex67 signal can be also seen in the Rsa4-FTP lane. (d) Comparison between Mex67 in vivo CRAC data (Tuck, A.C. et al., Cell. 154, 996-1009, 2013) (lower panel) and the in vitro Mex67 CRAC data (upper panel). Total number of hits (from the in vitro CRAC data) was plotted along with the published in vivo CRAC data against the relative position along the rDNA sequence. The Y-axis is not normalized, as the two different data sets were not analyzed at the same time. The two specific sites, where Mex67-Mtr2 was significantly crosslinked to the Yvh1 particle, are labeled with '5.8S hit' and 'P0 hit'.

Supplementary Figure 3 Cryo-EM sorting scheme of the Yvh1 pre-60S particle.

Cryo-EM sorting scheme of the pre-60S particles affinity-purified via Yvh1-FTP and the FSC curve of the final subpopulation. Pre-60S particles obtained were 3D classified using iterative multireference projection alignment. The final subpopulation was refined to a resolution of 7.4 Å (Fourier shell correlation cutoff of 0.5).

Supplementary Figure 4 Cryo-EM analysis of the Yvh1 pre-60S particle.

(a) Model of the Yvh1 phosphatase domain fit into the cryo-EM density of the Yvh1 pre-60S particle contacting ribosomal protein Rpl12. The phosphatase domain of Yvh1 was modeled on PDB ID: 2OUD (Tao, X. et al., Protein Sci. 16, 880-886, 2007); the model of Rpl12 was taken from PDB ID: 4V7F (Leidig, C. et al., Nat Commun 5 , 3491, 2014). (b) View on the position of Rpl10, based on the structure of a mature 60S subunit taken from PDB ID: 4V88 (Ben-Shem, A. et al., Science 334 , 1524-1529, 2011) into the density of the Yvh1 particle, revealing that ribosomal Rpl10 is absent in the Yvh1 particle. (c) Positions of the helices H68, H69 and H71 in the early Arx1 particle (blue) and a mature 60S subunit (dark red). Models of the complete ribosomal RNA (PDB ID: 4V7F and PDB ID: 4V88) were fit into the density of the Yvh1 particle. There are no distinct densities visible for H68 and H69, whereas H71 is located in its mature position. (d and e) View on the 5.8S rRNA region in the Yvh1 particle (d) with the Mex67 crosslink hits to helices H7, H9-H10 (red) as compared to the early Arx1 particle (e) (EMD-2528; Leidig, C. et al., Nat Commun 5 , 3491, 2014), where CRAC hits of Mtr4 (pink) and Nop53 (cyan) are indicated.

Supplementary Figure 5 In vitro and in vivo interaction of Yvh1 with the pre-60S nuclear export machinery.

(a) The different pre-60S ribosomal particles were affinity-purified using the indicated Flag-TEV-ProtA-tagged bait proteins (FTP), and SDS-PAGE (upper panel) and western blotting using the indicated antibodies (lower panel) was used to analyze the final Flag eluates. Asterisk (*) marks the position of the bait protein. Note that the western blot membrane probed with α-Mrt4 and α-Mex67 antibodies contained the marker lane (M), whereas the other western blot membranes had no marker between lane 4 and 5, and therefore were cut between lanes 4 and 5 for better figure arrangement. This figure corresponds to a related Figure 4a, but in this case the eluates were probed with additional antibodies (e.g. α-Nsa2, α-Nug2 and α-Nog1) (b) Quantification of the extent of recombinant Mex67-Mtr2 binding to the Yvh1 particle in the absence or presence of either Mrt4 or Rsa4. The Coomassie stained gel shown in Fig. 4b was scanned in LAS 4000 (GE). Using Image Quant TL software, the intensity of the Mex67 band (normalized with respect to the intensity of the corresponding Lsg1 band) was determined for each lane of the depicted gel (lower panel). The region of the Coomassie-stained gel used for image analysis (see Fig. 4b) is shown in a blow up (lower panel). Lsg1, Arx1 and Mex67 bands stained by Coomassie are labeled. The stars in lane 1, 4 and 7 mark the presence of Hsp70 proteins (as determined by mass spectrometry), which co-migrate with endogenous Mex67. (c) Efficient co-enrichment of Mex67 with Lsg1 pre-60S particles requires Yvh1. Lsg1-FTP was affinity-purified derived from either wild-type YVH1 or yvh1Δ strain and Flag eluates were analyzed by SDS-PAGE (upper panel) and western blotting using the indicated antibodies (lower panel). Two independent affinity-purifications of Lsg1-FTP from wild-type (WT) and yvh1∆ cells are shown.

Supplementary Figure 6 Mex67 recruitment and P0-stalk biogenesis.

(a) MEX67 and MRT4 exhibit epistatic genetic relationship. Yeast shuffle strains monitoring MEX67 MRT4 wild-type cells, mex67∆loop MRT4 single mutant, MEX67 mrt4Δ single mutant and mex67∆loop mrt4∆ double mutant were spotted in serial 10-fold dilutions onto 5-fluoroorotic acid plates. It was incubated at 23°C, 30°C and 35°C for 2 days. (b and c) Synergistically enhanced (se) growth defects in the yvh1∆ nmd3nes1∆ double mutant (b) or synthetic lethal growth phenotype in the yvh1∆ nup116∆ double mutant (c). It was grown at the indicated temperatures and for the indicated time on YPD (b) or 5-fluoroorotic acid plates (c). (d) P0 facilitates Mex67-Mtr2 release from the exported pre-60S subunit. Growth of a GAL::RPP0 yeast strain carrying Rpl24-FTP on galactose (YPG) and glucose (YPD) containing plate (upper panel). Growth curve of wild-type yeast strain carrying Rpl24-FTP (L24-FTP) in comparison to strain GAL::RPP0/Rpl24-FTP in YPD (glucose containing) medium (lower panel). At time point t0, the strains have been transferred from galactose to YPD medium, and growth was followed over time by measuring OD600. (e) Rpl24-FTP was affinity-purified from GAL::RPP0 cells grown in galactose medium (lane 1) and depleted for P0 for 6 hr. (lane 2) and 9 hr. (lane 3) by growth in glucose medium. The FLAG eluates of affinity-purified Rpl24-FTP were analyzed by SDS-PAGE and western blotting using the indicated antibodies. The position of Rpl3, Rpl4 and Rpl5, as well as 3xHA-Rpp0 is indicated.

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Sarkar, A., Pech, M., Thoms, M. et al. Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit. Nat Struct Mol Biol 23, 1074–1082 (2016). https://doi.org/10.1038/nsmb.3312

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