2.7 Å cryo-EM structure of human telomerase H/ACA ribonucleoprotein

Abstract

Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase is comprised of two distinct functional lobes tethered by telomerase RNA (hTR): a catalytic core, responsible for DNA extension; and a Hinge and ACA (H/ACA) box RNP, responsible for telomerase biogenesis. H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs, which is critical for the biogenesis of the spliceosome and ribosome. Much of our structural understanding of eukaryotic H/ACA RNPs comes from structures of the human telomerase H/ACA RNP. Here we report a 2.7 Å cryo-electron microscopy structure of the telomerase H/ACA RNP. The significant improvement in resolution over previous 3.3 Å to 8.2 Å structures allows us to uncover new molecular interactions within the H/ACA RNP. Many disease mutations are mapped to these interaction sites. The structure also reveals unprecedented insights into a region critical for pseudouridylation in canonical H/ACA RNPs. Together, our work advances understanding of telomerase-related disease mutations and the mechanism of pseudouridylation by eukaryotic H/ACA RNPs.

Fig. 1. Structure of human telomerase H/ACA RNP.

a Secondary structure of hTR. RNA domains (colored as indicated) are divided into the catalytic core and the H/ACA RNP. The binding of the H/ACA heterotetramers and TCAB1 on the 5’ and 3’ RNA hairpins is shown. b Domain architectures of H/ACA protein subunits. NTE, N-terminal extension; TruB, tRNA pseudouridine synthase B-like; PUA, pseudouridine synthase and achaeosine transglycosylase; CTE, C-terminal extension; and RBD, RNA-binding domain. c Schematic of the human telomerase holoenzyme. This work focuses on the H/ACA RNP, thus the catalytic core is greyed out. d 2.7 Å cryo-EM reconstruction of the telomerase H/ACA RNP (Supplementary Figs. 3, 4). The subunits are colored as in (b). e A ribbon representation of the telomerase H/ACA RNP with subunits colored as in (b).

Fig. 2. Cross dyskerin-dyskerin interactions.

a Domain architectures of the 5’ and 3’ dyskerin. The domain color scheme is used throughout this figure. b Structure of the 5’ and 3’ dyskerin in the telomerase H/ACA RNP in space-filling (left) and ribbon (right) representations. The 3’ hydrophobic cleft and helix 1 of the 5’ dyskerin are labeled. For clarity, only domains of the dyskerin subunits are shown. c Interactions between helix 1 of the 5’ dyskerin and the 3’ hydrophobic cleft formed by the 3’ dyskerin. d, e The electrostatic potential of the 3’ hydrophobic cleft formed by the 3’ dyskerin and helix 1 of the 5’ dyskerin, respectively. f Cross-dyskerin interaction at the 3’ hydrophobic cleft (also see Supplementary Fig. 4b). Hydrogen-bonds are shown as dashed blue lines. g Disease mutations at the inter-dyskerin interface shown in (f). Residues whose mutations are associated with dyskeratosis congenita, HH syndrome and sporadic cancer are highlighted as spheres.

Fig. 3. Enhancement of the inter-dyskerin interaction by the 3’ NOP10.

a Schematic of the telomerase H/ACA RNP. The 5’ and 3’ dyskerin and the 5’ and 3’ NOP10 are highlighted. The 3’ hydrophobic cleft is circled with a dotted line. b Close-up view of helix 1 of the 5’ dyskerin, the NTE, TruB and PUA domains of the 3’ dyskerin, and the C-terminus of the 3’ NOP10. c Interactions of the 3’ NOP10 C-terminus at the 3’ hydrophobic cleft (also see Supplementary Fig. 4a). Hydrogen-bonding and van der Waals interactions are shown as dashed blue and dashed yellow lines, respectively. d, e Comparison of the 3’ hydrophobic cleft of the 3’ dyskerin (d) with the analogous region at the 5’ dyskerin subunit (e).

Fig. 4. Dyskerin-hTR interface rationalizes disease mutations.

a Structure of the H/ACA domain of hTR. The structure is overlayed atop the H/ACA proteins. Conserved H, ACA, CAB, and BIO motifs are indicated. Unresolved nucleotides are shown as blue dashed lines. b Positioning of the H box of hTR in a cavity formed by the 5’ dyskerin. c Interactions between residue G373 of the H box and the H box binding motif (HBM) of the 5’ dyskerin as part of the CTE domain (also see Supplementary Fig. 4f). Stacking interactions are shown as dashed yellow lines. Domains of the 5’ dyskerin are colored as shown in the schematic. d Dyskeratosis congenita and HH syndrome disease mutations in the HBM of the 5’ dyskerin. Disease-associated residues are highlighted as spheres. e Positioning of the ACA box of hTR in a cavity formed by the 3’ dyskerin. The 3’ end of hTR (G450) reaches towards the 5’ dyskerin. f Interactions between the 5’ and 3’ dyskerin, and the 3’ end of hTR (also see Supplementary Fig. 4e). Hydrogen-bonding and stacking interactions are shown as dashed blue and dashed yellow lines, respectively. g Dyskeratosis congenita, HH syndrome, aplastic anemia, and bone marrow failure disease mutations in the region shown in (f). Disease-associated residues are highlighted as spheres.

Fig. 5. TCAB1 interaction with hTR and the 3’ NHP2.

a Secondary structure of the P8 stem-loop of hTR, with CAB and BIO box labeled. Watson-Crick-Franklin base pairs are indicated as solid lines. Non-Watson-Crick-Franklin base pairs are indicated as dots. b Interactions of TCAB1 with the BIO and CAB boxes of hTR and the 3’ NHP2. Circles indicate the three contact regions depicted in (c–e). Unresolved loops are shown as green dashed lines. c Close-up view of the β-hairpin loop of TCAB1, and its interactions with the P8 stem of hTR (also see Supplementary Fig. 4g). d Close-up view of the TCAB1-CAB box interaction. Certain nucleotides of the CAB and BIO boxes are transparent for clarity. e Close-up view of the interactions between the NHP2 interacting loop (NIL) of TCAB1 and the 3’ NHP2. Hydrogen-bonding and van der Waals interactions are shown as dashed blue and dashed yellow lines, respectively.

Fig. 6. Interactions at the 3’ dyskerin thumb loop.

a Interactions between the 3’ dyskerin thumb loop, hTR and the 3’ GAR1. The circle indicates the 3’ dyskerin thumb loop interaction depicted in (b, c). The eukaryote specific CTE of GAR1 is colored yellow (Supplementary Fig. 6b). b Close-up view of the interaction between the 3’ dyskerin thumb loop and hTR (also see Supplementary Fig. 4d). The side-chain of the catalytic aspartate (D125) is shown in the dyskerin active site. Hydrogen-bonding and van der Waals interactions are shown as dashed blue and dashed yellow lines, respectively. c Close-up view of the interactions between the base of the 3’ dyskerin thumb loop and the 3’ GAR1 (also see Supplementary Fig. 4c). d, e Comparison of the human 3’ dyskerin active site and thumb loop with the substrate-bound archaeal Cbf5 from Pyrococcus furiosus (P. furiosus) (PDB 3HAY [10.2210/pdb3HAY/pdb]), respectively. The active site and the catalytic aspartate residue are indicated. In the Cbf5 structure shown in (e), the pseudouridylated residue (ψ7) of the substrate RNA is positioned in the active site. In our structure shown in (d), the dyskerin active site is empty.

Fig. 7. Mimicry of substrate RNA binding by hTR.

a Secondary structure of the guide RNA (grey) in complex with the substrate RNA (pink) in an archaeal H/ACA RNP (PDB 3HAY [10.2210/pdb3HAY/pdb]) shown in (b). The PS1 and PS2 helices are shown with the 5’ and 3’ guide sequences respectively. The unpaired ‘UN’ dinucleotide is labeled. Watson-Crick-Franklin base pairs are indicated as solid lines. Non-Watson-Crick-Franklin base pairs are indicated as dots. b Structure of the guide (grey) and substrate (pink) RNAs in an archaeal H/ACA RNP (PDB 3HAY [10.2210/pdb3HAY/pdb]). The 5’ and 3’ guide sequences are colored in dark-grey. The substrate-guide duplexes (PS1, PS2) are indicated. c Secondary structure of the 3’ hairpin of the hTR H/ACA domain in our structure shown in (d). Parts of hTR that resemble the 3’ guide sequence and the substrate RNA are colored in dark-grey and in pink, respectively. d Structure of hTR on the 3’ dyskerin observed in our structure. Regions of hTR are colored as described in (c).

Fig. 8. 3D variability analysis (3DVA) reveals conformational heterogeneity at the 3’ dyskerin thumb loop.

a Comparison of the 3DVA conformations (State 1 and State 2). The arrow indicates the degree of rotation/movement in State 2 relative to State 1. b An overlay of State 1 and State 2. The circle indicates the variability around the 3’ dyskerin thumb loop, including the P7 and P8a stems of hTR. c Close-up view of the 3’ dyskerin thumb loop in a semi-closed conformation. Arrows indicate the movement of hTR and the thumb loop from the semi-closed conformation to the open conformation described in (d). d Close-up view of the dyskerin thumb loop resembling the open conformation. Atomic models shown in (c) and (d) come from high resolution reconstructions (Supplementary Fig. 2, 11) and are fitted into the low resolution 3DVA densities. e An overlay of the thumb loop interaction with the 3’ GAR1 in the open conformation (colored) and the semi-closed conformation (grey) (also see Supplementary Fig. 12f). Residues of the 3’ GAR1 hydrophobic surface are shown. Changes from the semi-closed to open conformation are indicated with an arrow. Certain residues of the 3’ dyskerin thumb loop are transparent for clarity. f–h Schematics of proposed pseudouridylation cycle in eukaryotic H/ACA RNPs. The cycle includes substrate loading (f), pseudouridylation at the transition state (g) and product release (h). The dyskerin thumb loop changes conformation from partially closed (f) to fully closed (g) to open (h). The extent of thumb loop opening during product release is unknown and hence indicated by a question mark (?). Proposed states observed for the telomerase H/ACA RNP are indicated.