Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN–pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.
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Cryo-EM maps for wt-TSEN and endoX-TSEN have been deposited in the EMDB under the accession codes EMD-28755 and EMD-26856, respectively. The atomic model for endoX-TSEN has been deposited in the PDB under accession code 7UXA. Mass spectrometry data have been deposited at MassIVE with the dataset identifier MSV000091153. Source data are available for crosslinking mass spectrometry data and all uncropped western blots. Source data are provided with this paper.
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We thank P. Tumbale and J. Rodriguez for their critical reading of this manuscript. We would like to thank R. Huang and A. Zeher for help with cryo-EM data collection. This work utilized the Krios at the NCI–NICE Cryo-EM Facility. This work was supported by the US National Institute of Health Intramural Research Program and the US National Institute of Environmental Health Sciences (ZIA ES103247 to R.E.S., 1ZIC ES102488 to J.G.W., 1ZIC ES103206 to L.J.D., 1ZIC ES102487 to R.M.P., 1ZI ES043010 to L.P., and 1ZIC ES103326 to M.J.B.). This work was also supported by the US National Institute of Health Extramural Research Program and the US National Institute of General Medical Sciences (R35-GM136435, to A.G.M. and 1K99-GM143534 to C.K.H.).
The authors declare no competing interests.
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Extended Data Fig. 1 WT-TSEN cryo-EM processing workflow.
a. A representative micrograph, of curated 9634 micrographs. b. 1,557,097 particles were picked from the 9,634 curated micrographs, 10 representative 2D classes are shown. Particles contained in good classes were used to generate c. ab-initio reconstructions, three of which were selected and further refined using d. heterogenous refinement. The particles contained in the best three classes were further filtered using e. 2D classification and f. heterogeneous refinement prior to two classes being further refined using g. homogenous refinement, with 2x binned particles. h. A final round of heterogenous refinement resulted in one 3D class which was i. refined to an estimated 4.38 Å using homogenous refinement. j. Refinement continued with another round of homogeneous refinement resulting in a resolution of 4. Å following the unbinning of the final 161,512 particles with an estimated resolution of 4.2 Å. k. A local refinement resulted in a final map with an estimated resolution of 3.9 Å.
Extended Data Fig. 2 EndoX-TSEN structure cryo-EM processing workflow.
a. A representative micrograph of 8095 curated micrographs. b. 570,104 particles were picked from the 8059 curated micrographs and used for 2D classification. Particles from the good classes were used to generate c. ab-initio reconstructions, two of which were selected and further refined using d. heterogenous refinement. e. The particles contained in the best class were binned and refined with homogenous refinement. f. The particles were then reextracted and unbinned and used for another round of g. homogenous refinement followed by h. non-uniform refinement and i. local refinement resulting in a final map with an estimated resolution of 3.28 Å. j. FSC of the model to map.
Extended Data Fig. 3 Example density for the TSEN complex.
Example densities for residues along helices from a. TSEN34, b. TSEN54, c. TSEN15, d. TSEN2, the β9-β9 interfaces for β-sheets for e. TSEN34, f. TSEN54, g. TSEN15, h. TSEN2 and density for the i. proximal base pair of the tRNA as well as the L10 loops for j. TSEN54, k. TSEN15, and the l. cation-π.
Extended Data Fig. 4 BS3 crosslinking of TSEN complexes.
SDS-Page gels showing the DMSO control and crosslinked samples for a. wt-TSEN complex and b. TSEN Complex and CLP1. c-h. MSMS spectra of CLP1 peptides crosslinked to other members of the complex. Panels c (m/z 822.4292) and d (466.4795 m/z) show MSMS spectra of peptides arising from CLP1 and TSEN2. Panels e (m/z 412.2311), f (m/z 739.0369), g (m/z 671.3516), and h (m/z 398.5568) show MSMS spectra arising from crosslinks from CLP1 and TSEN54. Fragment ions that arise from green colored peptides are shown in green, fragments arising from blue colored peptides are shown in blue, and unassigned ions shown in black.
Extended Data Fig. 5 Molecular dynamics simulations account for stable complexes during dynamics.
a. Root mean square deviations (RMSD) of individual proteins and the complex averaged over each run and averaged over the five runs. TSEN34 has the largest deviations while TSEN15 shows smallest deviations irrespective of the RNA binding. Standard deviations are shown in parenthesis. The reference (Complex I) was the starting Cryo-EM configuration. Complex (II) is without tRNA and Complex (III) is without TSEN15. b. Root mean square fluctuations of individual residues averaged calculated during the microsecond dynamics and averaged over all runs. Standard deviations are shown as error bars. c. Representative dynamic cross correlation matrices of the protein components from Complex (I).
Extended Data Fig. 6 TSEN54 is the anchor that mediates the interaction of CLP1 with the TSEN complex.
Overexpression (in HEK cells) and immunoprecipitations of the individual TSEN proteins (lanes 1–4 and lanes 7–10) or full TSEN complex (lanes 5 and 11) in the absence (lanes 1–5) and presence (lanes 7–11) of CLP1 reveals strong association between CLP1 and TSEN54 (lane 10) as well as the full TSEN complex (lane 11). The experiments were conducted as previously reported1 using CLP1-TEV-GFP.
Extended Data Fig. 7 Structural comparison of the TSEN and EndA active site residues.
a. Overlay of TSEN + tRNA (colored as before) and EndA + BHB (grey, PDBID:2GJW) showing overall similar RNA binding. b. Overlay of the active site residues for the 3′ splice site and the c. cation-π residues from TSEN2 shown. d. Overlay of the cation-π residues from TSEN34 and the e. TSEN2 active site residues for the 5′ splice site. f. tRNA cleavage assays of a broccoli RNA -aptamer containing pre-tRNA-ILE using samples immunoprecipitated via the mutants of the 3′ splice site (pulled down by a FLAG tag on TSEN2, except for the 34 mutant) or g. 5′ splice site (pulled down by a FLAG tag on TSEN34, except for TSEN2 active site mutant). TSEN proteins that weren’t the pull-down target had a MYC-tag. The data shown is one representative experiment of three biological replicates.
Supplementary Data Figs. 1–4
Source Data Extended Data Fig. 4
Source Data Extended Data Fig. 4
Raw mass spectrometry data.
Source Data Extended Data Fig. 6
Uncropped western blots.
Source Data Extended Data Fig. 7
Uncropped western blots and gels.
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Hayne, C.K., Butay, K.J.U., Stewart, Z.D. et al. Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex. Nat Struct Mol Biol (2023). https://doi.org/10.1038/s41594-023-00991-z