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Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM

Nature Structural & Molecular Biology volume 21, pages 95–102 (2014)Cite this article

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Abstract

The eukaryotic exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3′-to-5′ exo- and endo-RNase activities. RNA substrates have been shown to be recruited through the core to reach Rrp44's exo-RNase (EXO) site. Using single-particle EM and biochemical analysis, we provide visual evidence that two distinct substrate-recruitment pathways exist. In the through-core route, channeling of the single-stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative direct-access route, this conformational change does not take place, and the RNA substrate is visualized to avoid the core and enter Rrp44's EXO site directly. Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic exosome and indicate substrate-specific modes of degradation by this complex.

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Figure 1: Rrp44–exosome degrades RNA substrates differently than does Rrp44 alone.
Figure 2: Comparison and analysis of the RE-short and RE-long 3D models.
Figure 3: Analysis of the RE incubated with streptavidin-labeled RNAs (SA–RNAs).
Figure 4: RNA substrates with short 3′ single-stranded overhangs can be recruited to the exosome via direct-access routes.
Figure 5: Alternative RNA substrate–recruitment routes by the RE during the degradation process.
Figure 6: tRNA with short 3′-end overhang was degraded through the direct route.
Figure 7: A hypothetical model for the degradation routes of RNA substrates with 3′ single-stranded overhangs in the Rrp44–exosome.

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Acknowledgements

We thank J.S. Butler (University of Rochester) for providing the yeast strain of TAP–Rrp46 ΔRrp6; J.-L. Lei and Y. Xu for the EM support; J. Wang for EM image processing–scripting help; the Wang-group members at Yale University and Tsinghua University and the Sui-group members at Tsinghua University for their helpful discussions; and E. Nogales, S. Wolin and S. Sui for their helpful comments on the manuscript. We acknowledge the China National Center for Protein Sciences Beijing and “Explorer 100” cluster system of Tsinghua National Laboratory for Information Science and Technology for providing the facility support. This study was funded by the National Basic Research Program of China grant 2010CB912401 (to H.-W.W.) and the US National Institutes of Health (NIH) grant GM-102543 (to A.K.). H.-W.W. is supported as a principal investigator of the Tsinghua-Peking Joint Center for Life Sciences and as the awardee of Youth One-Thousand Talent Program by the State Council of China. J.-J.L. is supported as the awardee of a Gatan Scholarship. M.A.B. was partially supported by the NIH Molecular Biophysics Training grant GM-008267.

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Author notes

  1. Matthew A Bratkowski

    Present address: Present Address: Cecil and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.,

  2. Jun-Jie Liu, Matthew A Bratkowski and Xueqi Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Jun-Jie Liu, Chu-Ya Niu & Hong-Wei Wang

  2. Joint Graduate Program of Peking-Tsinghua-National Institute of Biological Science, Tsinghua University, Beijing, China

    Jun-Jie Liu & Hong-Wei Wang

  3. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA

    Matthew A Bratkowski & Ailong Ke

  4. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA

    Xueqi Liu

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Contributions

J.-J.L., X.L., C.-Y.N. and H.-W.W. performed EM and single-particle analysis. J.-J.L., M.A.B., X.L. and C.-Y.N. purified the exosome complex. M.A.B. and A.K. performed RNA degradation assays of wild-type and mutant complexes. J.-J.L., M.A.B., X.L., A.K. and H.-W.W. planned the experiments. J.-J.L., M.A.B., A.K. and H.-W.W. wrote the manuscript.

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Correspondence to Ailong Ke or Hong-Wei Wang.

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Integrated supplementary information

Supplementary Figure 1 The HDV-ribozyme RNA substrates and the RE complexes used in the biochemical assays.

(a) Schematics of the HDV ribozyme RNA with the key structural features and processing pausing sites highlighted. (b) List of the AU-rich 3' ss-overhang sequences in the HDV RNAs. (c) HDV RNAs before (−) and after (+) the snap-cooling refolding procedure, analyzed on an 8% native polyacrylamide gel (PAGE) and visualized by autoradiography. (d) Degradation assay of HDV RNA substrates containing different length of 3'-end overhang tails (0, 8, 20, 30, and 40 nt AU-rich sequences). R: RNA only control. RE: Rrp44-exosome reactions. Positions of intermediates and end products are marked by arrows and were determined by hydroxyl radical and RNase T1 ladders. (e) Degradation assay with Rrp44 active site mutants alone or in complex with the core exosome. All reactions were incubated for 90 minutes. The labels WT, EX-, EN-, and DM stand for wild-type, exonuclease mutant (D551N), endonuclease mutant (D117N), and double mutant (D551N D117N), respectively. (f) Mapping the pausing sites by Rrp44 and RE complex using the RNase T1 (cleaves after the G nucleotide) and alkaline hydrolysis ladders. (g) SDS-PAGE revealing the separation of free Rrp44 from RE using anion-exchange chromatography after the reconstitution of mutant Rrp44-exosome complexes. As indicated by the fraction tube numbers labeled to the bottom of the gel, the free Rrp44 peak is well separated from the RE peak, and stringent pooling of the RE fractions further improves the purity. Only the RE-D551N reconstitution is shown here; other reconstitutions followed an identical pattern. The wild-type RE was also purified in this way after the TAP-affinity purification to remove any free Rrp44 in the sample. (h) Typical purity of the TAP-Rrp46 purified wild-type RE complex analyzed by negative stain EM. The scale bar is 50 nm.

Supplementary Figure 2 Gel-shift assays.

Biotinylated RNA substrates interact with RE in the presence of 5 mM EDTA in the buffer (a) and with streptavidin in RNase free water (b). Note that RNA08 is not visible because it ran out of the gel. The gel was stained with ethidium bromide and imaged under UV.

Supplementary Figure 3 SA–RNA–RE interactions.

(a) HRP-SA-RNA pull down assay by exosome-bead. SA-RNA08, SA-RNA16, SA-RNA24 (lane1-3) showed low affinity for the exosome-bead, while SA-RNA36, SA-RNA47, SA-RNA50 (lane4-6) had a high affinity. Controls appear in lanes C1 (HRP-SA with empty beads), C2 (a mixture of six kinds of HRP-SA-RNAs), and C3 (HRP-SA alone). (b-c) Class average image with SA density in the SA-RNA47-RE sample (b) and the SA-RNA50-RE sample (c), respectively. The corresponding reprojection and surface view of RE-long model was placed on Row 2 and 3. The corresponding reprojection and surface view of RE-short were placed on Row 4 and 5. The dimension of each particle box is 40 nm. (d-e) Reconstructions of RE incubated with six types of SA-RNAs in the front view (d) and back view (e), respectively. SA-RNA08-RE (dark gray), SA-RNA16-RE (yellow), SA-RNA24-RE (cyan), SA-RNA36-RE (blue), SA-RNA47-RE (purple) and SA-RNA50-RE (pink) 3D volumes (about 20,000 particles were used for each sample). The additional density in SA-RNA47-RE and SA-RNA50-RE is marked with black arrow.

Supplementary Figure 4 Back-projection of 2D class averages to estimate streptavidin location in 3D.

(a) Based on the spatial location of SA defined by class-average and surface view, we used a rod perpendicular to the corresponding view of 2D class average to represent the SA projection on the 3D model of apo-RE. The points of most rod intersections (marked as orange spheres) provide estimation of a highly probable location of SA attached to the 3D RE volume. (b) As a positive control of the method, we get a definite location of SA attached to the top of SA-RNA47-RE complex, agreeing well with the 3D reconstruction.

Supplementary Figure 5 The distribution of particles in RE + RNA24–SA samples.

For each sample at every reaction time point, about 30,000 to 55,000 particles were used to do 2D alignment and classification for the statistics analysis. Error bars, defined by standard deviation, were calculated based on 3 individual particle sets for each sample. Only bottom dots (SA around Rrp44) are detected for these samples.

Supplementary Figure 6 HDV40 in complex with RE in the presence of 0.1 mM MgCl2.

The grey map is a 3D reconstruction from all the particle images taken of RE incubated with HDV40 in the presence of 0.1 mM MgCl2 at room temperature for 10 minutes. The Rrp44 region of the map is much weaker compared to the core. After supervised-classification using the two initial models, one as this grey model and the other as a low-pass filtered RE-long structure, the two outcome maps show clear RE-short (cyan) and RE-long (blue) conformations respectively.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–4 and Supplementary Note (PDF 4098 kb)

Supplementary Video 1

Docking of the atomic models in the 3D map of RE-short conformer. (MOV 15399 kb)

Supplementary Video 2

Docking of the atomic models in the 3D map of RE-long conformer. (MOV 15804 kb)

Supplementary Video 3

A morphing movie between the RE-short and RE-long conformers. (MOV 659 kb)

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Liu, JJ., Bratkowski, M., Liu, X. et al. Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM. Nat Struct Mol Biol 21, 95–102 (2014). https://doi.org/10.1038/nsmb.2736

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