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.

Fig. 1 ∣. RNase PNK Samples Multiple Conformational States.

a, Steps in the ITS2 pre-rRNA processing pathway that involve the RNase PNK components, Las1 and Grc3. Las1 cleaves at the C2 site leaving 2′-3′-cyclic phosphate (cP) and 5′-hydroxyl (OH) RNA ends followed by 5′-phosphorylation (P) by the Grc3 RNA kinase. Together this marks ITS2 for decay by the Rat1 exonuclease and the nuclear exosome. b, Size exclusion chromatogram of cross-linked (red) and non cross-linked (black) RNase PNK. Prior to gel filtration, BS3 (bis(sulfosuccinimidyl)suberate) cross-linker (50 μM) was incubated with 10 μM RNase PNK and incubated for 5 minutes at 22°C. Molecular weight (kDa) standards are indicated at the top. The gray bar corresponds to the peak fraction (Peak Frac.) shown on the SDS-PAGE gel which was used for cryo-EM and mass spectrometry analysis. Asterisks marks cross-linked RNase PNK species. Uncropped SDS-PAGE gel shown in Supplementary Data Set 1. c, BS3 cross-links identified within the RNase PNK complex. Purple arches represent intra- and inter-molecular Grc3-Grc3 and Las1-Las1 cross-links. Green lines represent inter-molecular Las1-Grc3 cross-links, while red arches represent inter-molecular Las1-Las1 cross-links assigned to the same lysine residue encoded within each Las1 protomer. d, Representative 2D classes of RNase PNK. e, Cryo-EM reconstruction (gray) and modeled RNase PNK determined in the absence of ligand (apo, yellow) and in the presence of ATP-γS (state 1, teal and state 2, purple). f, Superposition of apo (yellow), state 1 (teal) and state 2 (purple) RNase PNK ribbon diagrams. Double headed arrows mark the global displacement.

Fig. 2 ∣. Architecture of RNase PNK.

a, Domain architecture of C. thermophilum RNase PNK. Numbering above the diagram indicates the amino acid residue domain boundaries. Dashed lines indicate the proteolytic sensitive N-terminus of Grc3 that was removed prior to cryo-EM sample preparation. White boxes represent the CC domain of Las1 which was not visible in the structure. Colored stars mark the position of the catalytic centers. b, Apo cryo-EM reconstruction of RNase PNK with the individual domains colored as in 2a. c, Orthogonal views of the apo RNase PNK model shown as a cartoon and colored as in 2a. Las1 nuclease (RNase) and Grc3 kinase (PNK) active sites are highlighted in purple and red, respectively.

Fig. 3 ∣. RNase PNK Contains Dual RNA Binding Clefts.

a, Cryo-EM reconstruction of apo RNase PNK colored as in Figure 2a. The dashed circle marks the position of one of the symmetric RNA binding clefts. b, Surface representation of RNase PNK. The kinase active site is colored in red and the HEPN RNase site is colored in purple. Single stranded RNA (model RNA, cyan) was modeled into one of the RNA binding clefts using the coordinates from the RNA-bound crystal structure of Clp1 (PDB ID 4OHV). c, Electrostatic surface potential showing the RNA binding clefts (circled) formed at the interface of Las1 and Grc3. d, To determine whether both RNA binding clefts can be occupied by RNA simultaneously, we performed isothermal titration calorimetry (ITC) with S. cerevisiae RNase PNK and a minimal single-stranded RNA substrate (ssRNA) or a C2 cleaved RNA mimic of the S. cerevisieae ITS2 (SC-ITS2-cleaved-RNA). A representative ITC titration is presented along with the dissociation constant (K_d). The mean number of binding sites (N) and s.d. for ssRNA and SC-ITS2-cleaved-RNA were calculated from n=5 and n=3 replicates using independent protein preparations, respectively.

Fig. 4 ∣. Rearrangement of catalytic residue H142 within the Las1 HEPN nuclease site.

a, Ribbon diagram of the Las1 HEPN-HEPN dimer in state 1 and state 2. The conserved RϕxxxH motif responsible for Las1 RNA cleavage is purple and individual residues are shown as sticks. The boxes represent zoomed in views of the nuclease active site in state 1 and state 2. The table defines the equivalent RϕxxxH consensus residues in C. thermophilum Las1 (CtLas1) and S. cerevisiae Las1 (ScLas1). b, Amino acid sequence alignment of the HEPN RHxxxH motif from Las1 homologues. Chaetomium thermophilum (Ct), Saccharomyces cerevisiae (Sc), Gossypium arboretum (Ga), Noccaea caerulescens (Nc), Papilio machaon (Pm), Mizuhopecten yessoensis (My), Ceratitis capitata (Cc), Xenopus laevis (Xl), Mus Musculus (Mm), and Homo sapiens (Hs). c, Growth curves of S. cerevisiae tetO₇-LAS1 strain transformed with plasmids harboring wild-type Las1 (RHxxxH), Las1 RAxxxH variant (ScLas1 H130A), Las1 EHxxxA variant (ScLas1 R129E, H134A) and empty YCplac vector (vector). Strains were grown in the presence (light) or absence (dark) of 40 μg/ml doxycycline at 30°C. Each curve was generated by plotting the mean and s.d. calculated from n=3 independent experiments. d, Endogenous expression of Las1 variants and Grc3 in transformed S. cerevisiae tetO₇-LAS1 strain grown in YPD supplemented with 40 μg/ml doxycycline at 30°C. Cell lysate was analyzed by western blot using anti-Myc antibody (Grc3), anti-Flag (Las1) and anti-tubulin (loading control). Representative blots from n=2 independent experiments. e, Polysome profile of transformed tetO₇-LAS1 strain grown in the presence of 40 μg/ml doxycycline at 30°C. Black arrows mark ribosome half-mers, which is a hallmark for defective ribosome maturation. Representative profiles from n=3 independent experiments. f, Northern blot analysis of transformed S. cerevisiae tetO₇-LAS1 strains grown in YPD supplemented with 40 μg/ml doxycycline at 30°C. RNA loading was monitored using the 18S mature rRNA and SCR1. The integrative density of 27S pre-rRNA was normalized to 18S rRNA and reported below the blot. Representative blots from n=3 independent experiments. g, Cartoon of ITS2 mimicking RNA substrate (Sc-ITS2-RNA) used for in vitro C2 cleavage assays (left). Denaturing gels of C2 cleavage activity of ScRNase PNK variants (0-0.8 μM) incubated with Sc-ITS2 RNA (50 nM) (middle). C2 cleavage was quantified from n=3 technical replicates and the mean and s.d. were plotted (right). Specific activity normalized to wild-type ScRNase PNK C2 cleavage is reported. Source data for 4c, 4e, and 4g are available online. Uncropped images are shown in Supplementary Data Set 1.

Fig. 5 ∣. The Las1 RNase activity is uncoupled from ATP.

a, Vector map depicting the displacement of corresponding Cα atoms in ATP-γS bound RNase PNK states 1 and 2. Vector lengths correspond to domain motion. ATP-γS is shown as sticks. The metal, which is most likely magnesium, is shown as a black sphere. b, RNA cleavage using an ITS2 RNA mimic substrate (SC-ITS2-RNA; 0.1 μM) with recombinant RNase PNK (0.8 μM) in the absence (no nucleotide) and presence (ATP, gray) of purified 2 mM ATP. The mean and s.d. were blotted from n=3 technical replicates. Source data for 5b is available online.

Fig. 6 ∣. Conformational Changes within the RNase PNK Active Sites are Coordinated.

Local rearrangements within the nuclease and kinase active sites for the apo state, state 1 and state 2. Superposition of state 1 (color) and state 2 (gray) were generated by aligning Las1 α6 and highlights defined side chain rearrangements. In state 1, Las1 H142 is oriented towards the HEPN nuclease site (RNase site) while Grc3 W400 is arranged to π-stack with incoming RNA in state 2.

Fig. 7 ∣. Model of ITS2 processing by RNase PNK.

RNase PNK binds the ITS2 encoded pre-60S ribosome to initiate ITS2 processing. Global conformational changes to RNase PNK are coupled with local changes in the nuclease and kinase active sites driving ITS2 processing. RNase PNK adopts a spectrum of conformations including an extended state (Nuclease Active, state 1) where the catalytic Las1 H142 of RHxxxH points towards the nuclease active site for C2 cleavage of the ITS2 leaving 2′-3′ cyclic phosphate and 5′-hydroxyl (OH) RNA ends. Additional conformational changes reposition the 5′-hydroxyl RNA into one of the Grc3 RNA kinase active sites. Through a contracted conformation (Kinase Active, state 2), the catalytic W400 of the Grc3 kinase site adopts an alternative position to π-stack with an incoming RNA for phosphorylation (P). This coordinated process primes the 26S rRNA for processing by the Rat1-Rai1 exonuclease and the 7S rRNA for targeted RNA degradation by the nuclear exosome.