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Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

Nature Structural & Molecular Biology (2023)Cite this article

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Abstract

Heterotetrameric human transfer RNA (tRNA) splicing endonuclease TSEN catalyzes intron excision from precursor tRNAs (pre-tRNAs), utilizing two composite active sites. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH). Despite the essential function of TSEN, the three-dimensional assembly of TSEN–CLP1, the mechanism of substrate recognition, and the structural consequences of disease mutations are not understood in molecular detail. Here, we present single-particle cryogenic electron microscopy reconstructions of human TSEN with intron-containing pre-tRNAs. TSEN recognizes the body of pre-tRNAs and pre-positions the 3′ splice site for cleavage by an intricate protein-RNA interaction network. TSEN subunits exhibit large unstructured regions flexibly tethering CLP1. Disease mutations localize far from the substrate-binding interface and destabilize TSEN. Our work delineates molecular principles of pre-tRNA recognition and cleavage by human TSEN and rationalizes mutations associated with PCH.

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Fig. 1: Structure of TSEN–pre-tRNA complexes.
Fig. 2: Interaction networks between TSEN subunits and pre-tRNA substrate.
Fig. 3: Molecular determinants of 3′ splice site selection.
Fig. 4: Structure-guided assessment of PCH-associated mutations and effects on CLP1 binding.

Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-14925 (full-length TSEN–pre-tRNATyrGTA complex) and EMD-14923 (truncated TSEN–pre-tRNAArgTCT complex). Atomic coordinates of truncated TSEN–pre-tRNAArgTCT complex were deposited to the Protein Data Bank (http://www.rcsb.org) under accession number 7ZRZ. MD simulation datasets were uploaded to the Zenodo Open Science repository (https://doi.org/10.5281/zenodo.6513519). Reference models were derived from the AlphaFold Protein Structure Database with identifiers AF-Q8NCE0-F1, AF-Q8WW01-F1, AF-Q9BSV6-F1, and AF-Q7Z6J9-F1, or retrieved from the protein data bank (PDB) with accession codes 6GJW, 6Z9U, 3L0U, and 1EHZ. All data are available in the main text, public repositories, or the supplementary materials. Source data are provided with this paper.

Code availability

All codes used in this study are freely available to academic users via the indicated resources.

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Acknowledgements

We thank P. Devant, T.J. Heinke, and L. Sagert, Institute of Biochemistry, Goethe University Frankfurt, for biochemical characterization of TSEN and support in cell culture. We are grateful to T. Gewering and A. Möller for initial single-particle EM analyses and C. Kraft for technical support during cryo-EM data collection. Electron cryo-microscopy was carried out in the cryo-EM-facility of the Julius-Maximilians University Würzburg, and the cryo-EM infrastructure of the Institute of Biochemistry, Goethe University Frankfurt, and Collaborative Research Center 1507 (Z02–high-resolution cryo-EM platform). We thank S. Weitzer and J. Martinez for critical reading of the manuscript. This study was supported by the Collaborative Research Center 902 ‘Molecular Principles of RNA-Based Regulation’ and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – INST 161/977-1 FUGG. S.S. acknowledges a Boehringer Ingelheim Fonds fellowship. R.T. acknowledges funding by the European Research Council (ERC Advanced Grant No. 789121) and the German Research Foundation (Grant No. TA157/15-1). S.T. acknowledges funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – TR 1711/1-1.

Author information

Authors and Affiliations

  1. Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany

    Samoil Sekulovski, Lukas Sušac, Robert Tampé & Simon Trowitzsch

  2. Faculty of Biology, Johannes Gutenberg University Mainz, Mainz, Germany

    Lukas S. Stelzl

  3. KOMET 1, Institute of Physics, Johannes Gutenberg University Mainz, Mainz, Germany

    Lukas S. Stelzl

  4. Institute of Molecular Biology (IMB), Mainz, Germany

    Lukas S. Stelzl

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  1. Samoil Sekulovski
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Contributions

S.S. produced and purified protein–RNA complexes, performed biochemical assays, and prepared cryo-EM grids. L.S. prepared and screened cryo-EM grids, collected cryo-EM data, and guided data processing carried out by S.S. and S.T. S.S. and S.T. built the atomic model. L.S.S. performed all-atom molecular dynamics simulations. S.S. and S.T designed the experiments and wrote the initial draft of the manuscript with input from all authors. R.T. acquired funding. S.T. conceived the project, acquired funding, and supervised the work.

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Correspondence to Simon Trowitzsch.

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Nature Structural & Molecular Biology thanks Anita Hopper and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Biochemical and biophysical properties of TSEN/pre-tRNA complexes.

a, Size exclusion chromatograms of TSEN/pre-tRNATyrGTA complex and single components. Migration profile on a Superdex 200 Increase 3.2/300 column of TSEN/pre-tRNATyrGTA complex (black), TSEN only (cyan) and pre-tRNATyrGTA only (grey). b, SDS-PAGE analysis of truncated TSEN (TSENcore) (n = 3; independent biological replicates). c, Endonuclease activity assay. Yeast pre-tRNAPheGAA was incubated without (-) and with TSENcore and analyzed by urea-PAGE (n = 2; independent biological replicates). d, Endonuclease activity assay with full-length TSEN and TSENcore. Pre-tRNAArgTCT or pre-tRNATyrGTA was incubated without (-) and with full-length and truncated TSEN complex as indicated and analyzed by urea-PAGE (n = 3; independent biological replicates). e, Simulated flexibility of TSEN/pre-tRNAArgTCT. A hybrid model of truncated TSEN/pre-tRNAArgTCT with the central TSEN2 domain (residues 1-143 and 217-459) from the AlphaFold structure prediction33 and in silico modeled intron bases 37 to 44 was subjected to all-atom molecular dynamics simulations for assessment of structural flexibility. Color-coding is according to per residue B factor (Å2) with flexible regions in red and rigid regions in blue. The transparent local resolution filtered map of the complex is superimposed on the model.

Source data

Extended Data Fig. 2 Cryo-EM data processing workflow of human TSEN (detailed in Methods).

a,b,e, Representative micrographs of two TSEN/pre-tRNATyrGTA datasets (a,b) (1,135 and 1,096 micrographs, respectively) and truncated TSEN/pre-tRNAArgTCT (8,611 micrographs) (e). White size markers indicate 50 nm. c,f, Workflow of cryo-EM data processing for TSEN/pre-tRNATyrGTA (c) and truncated TSEN/pre-tRNAArgTCT (f) with final map and resolution. d,g, Representative 2D class images of TSEN/pre-tRNATyrGTA (d) and TSEN/pre-tRNAArgTCT (g). White size markers indicate 10 nm. h, Heterorefined ab initio models of TSEN/pre-tRNAArgTCT. as shown in (f). Percentages indicate particle distribution among maps. The model in dotted box was further refined by non-uniform refinement.

Extended Data Fig. 3 Quality of cryo-EM maps.

a,b, Gold-standard Fourier shell correlation curves, local resolution maps, and Azimuth plots of TSEN/pre-tRNATyrGTA (a) and TSEN/pre-tRNAArgTCT (b). Resolution values are derived from corrected curves without FSC-mask auto-tightening. The same color key was used for both local resolution maps. c-g, Representative cryo-EM densities enclosing the atomic models of pre-tRNAArgTCT secondary and tertiary structure base pairs (c), TSEN subunits (d), 3’ bulge cation-π sandwich (e), the 3’ splice site (f), and an interaction of TSEN2 N413 with the G-U wobble base pair of the A-I helix (g).

Extended Data Fig. 4 Structural aspects of TSEN/pre-tRNA complexes.

a, Surface representation of TSEN54 and TSEN34 highlight their common N-terminal domains (NTD, cartoon representation). CTD – C-terminal domain, NTE – N-terminal extension. b, Superposition of E. coli tRNAPheGAA (cyan; PDI ID 3L0U) onto human pre-tRNAArgTCT (grey, this work).

Extended Data Fig. 5 Electrostatic interactions of human and A. fulgidus splicing endonuclease.

a, Electrostatic surface potential of human TSEN with pre-tRNA (grey). b, Electrostatic surface potential of Archaeoglobus fulgidus endonuclease (PDB ID 2GJW) with BHB-RNA (grey). For comparison, the Archaeal endonuclease structure was superimposed with the cryo-EM model of TSEN-pre-tRNAArgTCT.

Extended Data Fig. 6 Molecular details of TSEN interactions in regions of PCH mutation hotspots.

a, TSEN54 (green) S85 environment in proximity to pre-tRNA acceptor stem (grey). b, Environment of the TSEN54 Y119D mutation potentially impacting pre-tRNA (grey) recognition. c, Environment of the TSEN15 (yellow) W76 environment. TSEN34 is shown in slate blue. d, TSEN15 Y152 environment.

Supplementary information

Source data

Source Data Fig. 4

Unprocessed gel.

Source Data Extended Data Fig. 1a

Table for size exclusion chromatography runs.

Source Data Extended Data Fig. 1

Unprocessed gel.

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Sekulovski, S., Sušac, L., Stelzl, L.S. et al. Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN. Nat Struct Mol Biol (2023). https://doi.org/10.1038/s41594-023-00992-y

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