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Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex

Abstract

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.

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Fig. 1: RNase PNK samples multiple conformational states.
Fig. 2: Architecture of RNase PNK.
Fig. 3: RNase PNK contains dual RNA binding clefts.
Fig. 4: Rearrangement of catalytic residue H142 within the Las1 HEPN nuclease site.
Fig. 5: The Las1 RNase activity is uncoupled from ATP.
Fig. 6: Conformational changes within the RNase PNK active sites are coordinated.
Fig. 7: Model of ITS2 processing by RNase PNK.

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Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-20042 (apo), EMD-20041 (ATP-γS bound state 1), and EMD-20040 (ATP-γS bound state 2). Atomic coordinates have been deposited in the Protein Data Bank under accession codes PDB 6OF4 (apo), PDB 6OF3 (ATP-γS bound state 1), and PDB 6OF2 (ATP-γS bound state 2).The MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014738. Source data for Figs. 4c,e,g and 5b are available with the paper online. All other data will be made available upon reasonable request.

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Acknowledgements

We thank T. Hall, W. Beard and M. Schellenberg, as well as all of the members of the Stanley Lab for their critical reading of this manuscript. We are grateful to all the members of the Molecular Microscopy Consortium for their help with cryo-EM data collection and processing. We thank L. Deterding from the NIEHS Mass Spectrometry Research and Support Group for help with cross-linking MS analysis. This work was supported by the US National Institute of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS; ZIA ES103247 to R.E.S.; ZIC ES103326 to M.J.B.) and the Canadian Institutes of Health Research (CIHR; 146626 to M.C.P.).

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Authors

Contributions

M.C.P. and R.E.S. conceived and designed all studies and wrote the manuscript, which was edited and approved by all authors. Cryo-EM data collection, processing, and refinement was carried out by M.C.P., A.L.H., M.J.B., J.M.K., and R.E.S. MS were carried out by M.C.P. and J.G.W. All other experiments were performed by M.C.P., K.H.G., and M.S.

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Correspondence to Robin E. Stanley.

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Integrated supplementary information

Supplementary Figure 1 Overview of the cryo-EM data processing scheme for RNase PNK.

a, A representative micrograph of cross-linked RNase PNK in vitreous ice and the corresponding power spectrum collected on a Titan Krios. b, 1016 micrographs of RNase PNK were collected from an UltrAuFoil R1.2/1.3 300 mesh grid. Picked particles (761,763) were subjected to 3D classification followed by 3D refinement in RELION (Scheres, S.H., J. Struct. Biol. 180, 519–530, 2012) prior to importing the particle stack into cisTEM (Grant, T. et al., Elife. 7, e35383, 2018). Additional 3D classification and refinement resulted in a C2-symmetric class of 102,753 particles refined to 3.2 Å resolution. c, Angular distribution of RNase PNK particles. d, Fourier shell correlation (FSC) curve for the RNase PNK reconstruction. The overall resolution is 3.2 Å according to the FSC 0.143 criteria (Scheres, S.H. et al., Nat. Methods. 9, 853-854, 2012; Rosenthal, P.B. et al., J. Mol. Biol. 333, 721–745, 2003). e, Cryo-EM reconstruction of RNase PNK colored based on local resolution calculated using MonoRes (Vilas, J.L. et al., Structure. 26, 337–344, 2018) and shown on the complete volume with two different orientations. f, Representative cryo-EM density for regions of Las1 and Grc3 that are highlighted in the text such as the Las1 HEPN motif (purple). Helices are colored as shown in Fig. 2.

Supplementary Figure 2 Overview of the cryo-EM data processing scheme for ATP-γS bound RNase PNK.

a, A representative micrograph and power spectrum of cross-linked RNase PNK associated with ATP-γS collected on a Titan Krios. b, 833 micrographs of ATP-γS bound RNase PNK were collected from an UltrAuFoil R1.2/1.3 300 mesh grid. Picked particles (703,078) were subjected to 3D classification followed by 3D refinement in RELION (Scheres, S.H., J. Struct. Biol. 180, 519–530, 2012) prior to importing the particle stack into cisTEM (Grant, T. et al., Elife. 7, e35383, 2018). Without imposing symmetry, additional 3D classification was performed to identify asymmetric classes that could be indicative of RNase PNK bound to RNA. Unfortunately, we did not observe asymmetric classes nor unassigned density among any of the 12 classes that could account for the presence of RNA. This observation is in line with the weak RNA binding affinity of RNase PNK (see Fig. 3d) and the low sample concentration used during cryo-EM grid preparations. For these reasons, we performed a subsequent 3D refinement on the most populated classes while imposing C2 symmetry to generate ATP-γS bound state 1 comprised of 74,582 particles and refined to 3.0 Å resolution along with state 2 comprised of 70,561 particles and refined to 2.9 Å resolution. c, Angular distribution of ATP-γS bound RNase PNK particles. d, Fourier Shell Correlation (FSC) curves for the ATP-γS bound RNase PNK reconstructions. The overall resolution for state 1 and state 2 are 3.0 Å and 2.9 Å, respectively, based on the FSC 0.143 criteria (Scheres, S.H. et al., Nat. Methods. 9, 853-854, 2012; Rosenthal, P.B. et al., J. Mol. Biol. 333, 721–745, 2003). e, Cryo-EM reconstructions of state 1 and state 2 colored to local resolution calculated using MonoRes (Vilas, J.L. et al., Structure. 26, 337–344, 2018). f, Cryo-EM density of the Grc3 ATP binding pocket from the apo dataset (empty pocket) compared to the ATP pocket for states 1 and 2 which contain ATP-γS (cyan) and Mg2+ (green sphere).

Supplementary Figure 3 Comparison of the Las1 HEPN domain structure in the three different cryo-EM states.

a, Ribbon diagram of the apo Las1 HEPN nuclease domain (orange) with secondary structure elements indicated and the conserved RϕxxxH nuclease motif in purple. b, Superimposition of the RNase PNK HEPN protomer from states 1 (color) and 2 (gray). State 1 Las1 RHxxxH motif is shown in purple and the loop (residues 115-124) between α4 and α5 is in blue. The coordinated conformational rearrangement of Met 124 and H142 are highlighted using arrows. Dotted line marks the loop in state 2 that is disordered. c, Two views of the cryo-EM density (gray) of the apo state, state 1 and state 2 Las1 HEPN nuclease active site.

Supplementary Figure 4 Comparison of the eukaryotic RNA specific polynucleotide kinases Grc3/Clp1 and RNase PNK interfaces.

a, Cartoon and ribbon diagrams of Grc3 and Clp1 (PDB ID 4OHV; Dikfidan, A. et al., Mol. Cell. 54, 975-986, 2014) with the domains colored as indicated. The residue numbers for Grc3 are from C. thermophilum and the residue boundaries for Clp1 are from C. elegans. Grc3 and Clp1 are composed of three domains including the central PNK domains that are structurally similar and flanked by distinct N- and C-terminal domains (NTD, CTD). b, The Grc3 CTD-Las1 interface with an inset depicting the Las1 LCT (Las1 C-terminal Tail)-Grc3 CTD interaction. The LCT is characterized by several well conserved hydrophobic residues including W347, W355, and I360 and it forms part of a β-sandwich with the Grc3 CTD. c, The Grc3 PNK-Las1 HEPN interface. The Grc3 PNK and Las1 RNase active sites are shown in red and purple, respectively.

Supplementary Figure 5 Grc3 nucleotide binding pocket.

State 1 and state 2 Grc3 ATP binding pockets occupied by an ATP analog (ATP-γS, cyan) and a metal ion, which is most likely magnesium (Pillon, M.C. et al., RNA. 24, 721-738, 2018) shown as a black sphere. Conserved Grc3 kinase motifs, such as the P-loop, Walker B, Clasp, and Lid motifs, essential for RNA phosphorylation are shown in red.

Supplementary Figure 6 Comparison of HEPN domain structures.

a-e, Ribbon diagram of the HEPN (orange)-HEPN (yellow) interface of several HEPN nucleases including a, Las1, b, Ire1 (PDB ID 2RIO; Lee, K.P. et al., Cell. 132, 89-100, 2008), c, RNase L (PDB ID 4O1P; Huang, H. et al., Mol. Cell. 53, 221-234, 2014), d, Cas13a (PDB ID 5XWP; Liu, L. et al., Cell. 170, 714-726, 2017) and e, MNT-toxin (PDB ID 5YEP; Jia, X. et al., J. Biol. Chem. 293, 6812-6823, 2018). Abbreviations N and C denote the N- and C-termini. While the secondary structure of the HEPN cores is largely conserved amongst HEPN nucleases, their tertiary and quaternary structural arrangement vary. The catalytic nuclease residues from the RϕxxxH motif (purple) are shown as sticks. The boxes represent a zoom of the RϕxxxH motif. The distance between Cα atoms encoded within the RϕxxxH motif are variable across the HEPN nuclease family. The HEPN domains of Las1 most closely resemble the HEPN domains from the bacterial MNT-toxin with a Dali score of 6.7 (Holm, L. et al., Nucleic Acids Res. 44, W351-W355, 2016). f, Surface representation of HEPN nucleases RNase PNK, Ire1 (PDB ID 2RIO) and RNase L (PDB ID 4O1P). HEPN-HEPN dimers are colored in orange with the juxtaposed RϕxxxH motifs highlighted in purple. The Grc3 polynucleotide kinase, Ire1 kinase and RNase L pseudo-kinase domains are shown in blue. Enzyme-specific insertions of RNase PNK, Ire1 and RNase L are colored in gray, brown and beige, respectively. The structural organization of the RNase PNK HEPN nuclease and polynucleotide kinase domains is distinct from Ire1 and RNase L, likely reflecting its unique enzymatic-coupling for RNA processing.

Supplementary Figure 7 Comparisons of RNase PNK active sites within ATP-γS bound state 1 and state 2.

To uncover the local structural changes within RNase PNK, we aligned states 1 and 2 using the Las1 RϕxxxH motif. Residue H142 encoded within the Las1 RHxxxH motif has alternative conformations between states 1 and 2, where H142 points towards the nuclease active site in state 1 and points towards the RNA binding cleft in state 2. RNA was modeled into the Las1 RNase site using the coordinates from the RNase L RNA engaged structure (PDB ID 4OAV; Han, Y. et al., Science. 343, 1244-1248, 2014). Alignment of the RNase L and Las1 RϕxxxH motifs uncovered the close proximity of Las1 H142 to the model RNA in state 1 suggesting this state is primed for RNA cleavage. The Grc3 Clasp residue W400 also has alternative conformations between states 1 and 2. RNA was modeled into the Grc3 PNK active site using the coordinates from the Clp1 RNA engaged structure (PDB ID 4OHY; Dikfidan et al. Mol. Cell. 54, 975-986, 2014). Alignment of the Clp1 and Grc3 PNK domains suggest that W400 is primed for π-stacking with the 5′-base of an incoming RNA substrate in state 2.

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Supplementary Information

Supplementary Figures 1–7, Supplementary Tables 1 and 2, Supplementary Notes 1 and 2

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Supplementary Data Set 1

Uncropped images from main text figures. Boxed regions highlight the areas used to generate the cropped images

Supplementary Video

Cryo-EM reconstruction of state 1. Model and cryo-EM map overlay showing overall quality.

Supplementary Video

Cryo-EM reconstruction of state 2. Model and cryo-EM map overlay showing overall quality.

Supplementary Video

Grc3-Las1 wing motions between ATP-γS bound state 1 and state 2. Morph between the ATP-γS bound RNase PNK states 1 and 2 highlights the side chain rearrangements within the RNase and PNK active sites

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Pillon, M.C., Hsu, A.L., Krahn, J.M. et al. Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex. Nat Struct Mol Biol 26, 830–839 (2019). https://doi.org/10.1038/s41594-019-0289-8

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