Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Sep 28;12(1):5610.
doi: 10.1038/s41467-021-25870-3.

Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Samoil Sekulovski #  1 , Pascal Devant #  1   2   3 , Silvia Panizza #  4 , Tasos Gogakos  5 , Anda Pitiriciu  1 , Katharina Heitmeier  1 , Ewan Phillip Ramsay  6 , Marie Barth  7 , Carla Schmidt  7 , Thomas Tuschl  5 , Frank Baas  8 , Stefan Weitzer  4 , Javier Martinez  9 , Simon Trowitzsch  10
Affiliations

Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Samoil Sekulovski et al. Nat Commun. .

Abstract

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Assembly and catalysis of recombinant human TSEN.
a Bar diagrams of TSEN subunits (TSEN15, green; TSEN34, orange; TSEN2, red; TSEN54, blue) and CLP1 (brown) depicting positions of PCH mutations, predicted nuclease domains of TSEN2 and TSEN34, and the RNA kinase domain of CLP1. The total amino acids of each protein are indicated. b SDS-PAGE of purified recombinant TSEN and TSEN/CLP1 complexes visualized by InstantBlue staining. Protein identities and size markers are shown. c Native mass spectrum of tetrameric TSEN complex from an aqueous ammonium acetate solution. Charge states of the predominant TSEN2–15–34–54 heterotetramer (blue circles), Heat Shock Protein (HSP) 70 (gray circles), the heterodimer TSEN15–34 (red circles), and TSEN15 (green circles) are indicated. d Pre-tRNA cleavage assay using tetrameric TSEN complex with Saccharomyces cerevisiae (S.c.) pre-tRNAPheGAA and mature tRNAPheGAA. Input samples and cleavage products were separated via urea-PAGE and visualized by Toluidine blue. RNA denominations are given on the right. e Pre-tRNA cleavage assay with TSEN heterodimers and S.c. pre-tRNAPheGAA. SDS-PAGE of the indicated heterodimers and the reconstituted TSEN tetramer is shown on the left (InstantBlue stain), urea-PAGE of the cleavage products on the right (Toluidine blue stain). Gels are representative of three independent experiments. Source data for b, d, and e, are provided as Source Data files.
Fig. 2
Fig. 2. Active involvement of the A–I base pair in coordinating pre-tRNA cleavage.
a Pre-tRNA cleavage assay comparing recombinant, inactive TSEN tetramer (TSEN2H377A and TSEN34H255A double mutant) to the TSEN/CLP1 complex. Cleavage products are visualized by denaturing urea-PAGE with subsequent Toluidine blue staining. RNA size markers are indicated on the left of the gel. b Pull-down assay with fluorescently labeled S.c. pre-tRNAPheGAA and inactive, tetrameric TSEN captured on protein G agarose functionalized with an α-His-antibody. Protein size markers are indicated on the left of each immunoblot, protein and RNA identities on the right. Input and co-precipitated, labeled pre-tRNAs were visualized by in-gel fluorescence, TSEN subunits, and the immunoglobulin G (IgG) heavy chain by immunoblotting. The IgG heavy chain served as a loading control. c Thermodynamic binding parameters of fluorescently labeled S.c. pre-tRNAPheGAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. d Thermodynamic binding parameters of fluorescently labeled tRNAPheGAA and inactive, tetrameric TSEN revealed by fluorescence anisotropy. Data in c and d are presented as mean values ± SD. e Schematic depiction of a pre-tRNA molecule showing ribonucleotides belonging to the mature domain (gray spheres), the intronic region (white spheres), the anticodon (black spheres), and the A–I base pair (yellow spheres). Proposed 5′ and 3′ splice sites (ss) are indicated. f Impact of A–I base pair mutations in S.c. pre-tRNAPheGAA on the endonucleolytic activity of tetrameric TSEN revealed by a pre-tRNA cleavage assay. C32:G54 – canonical A–I base pair, C32:C54 – disrupted A–I base pair, G32:C54 – inverted A–I base pair. All experiments are representatives of three independent assays. Source data for a, b, and f, are provided as Source Data files.
Fig. 3
Fig. 3. Structural and functional details of the TSEN15–34 dimer interface.
a X-ray crystal structure of a TSEN15–34 complex derived from limited proteolysis experiments. TSEN15 (green) and TSEN34 (orange) are shown in cartoon representation. Key amino acids are depicted in stick representation together with amino- (N) and carboxy (C)-termini. b Superposition of the TSEN15–34 heterodimer and the pre-tRNA endonuclease from Archaeoglobus fulgidus (AF) (PDB ID 2GJW). The position of the catalytic triad of TSEN34 (active site), the L10 loop of TSEN15, and the β-strands involved in the β-β interaction between TSEN15 and TSEN34 are shown. c Cartoon representation of the dimer interface with amino acid residues in stick representation (color coding as in a). Water molecules and hydrogen bonds are shown as red spheres and black dashed lines, respectively. d Cartoon representation of the TSEN15–34 interface highlighting histidine 116 (His116) of TSEN15, mutated in patients with a PCH type 2 phenotype. e Pull-down experiment with TSEN34, wt TSEN15, and TSEN15 carrying the H116Y mutation. Input and co-precipitated proteins were separated by SDS-PAGE and visualized by immunoblotting. Size markers and protein identities are shown. Blots are representative of two independent experiments. f Pre-tRNA cleavage assay with wt, tetrameric TSEN complex, and a tetrameric TSEN complex carrying the TSEN15H116Y (T15H116Y) mutation. Cleavage products were separated by urea-PAGE and visualized with toluidine blue. Experiments are representative of two independent assays. g Thermal stability of wt, tetrameric TSEN (black line), and TSEN15H116Y mutant complex (red line) assessed by differential scanning fluorimetry (DSF). Note that recombinant complexes were purified from HEK293 cells. Normalized (norm.) fluorescence is plotted against temperature in degree Celsius (°C). Denaturation temperatures (Td) of the complexes were derived from sigmoidal Boltzmann fits (gray dashed lines) with an error of fits. Standard deviations (SD) of technical triplicates are represented by red (T15H116Y) and black (wt) dashed lines (P = 0.001; unpaired, two-tailed Student´s t-test). Source data for e and f are provided as Source Data files.
Fig. 4
Fig. 4. PCH mutations affect thermal stability but not activity of recombinant TSEN.
a Immunoblot analysis of a pull-down assay with wt and mutant TSEN complexes co-expressed in HEK293 cells. Size markers and protein identities are indicated. b SDS-PAGE of SEC peak fractions of purified recombinant wt and mutant heterotetrameric TSEN complexes. c Pre-tRNA endonuclease assay of radioactively labeled S.c. pre-tRNAPheGAA with increasing amounts of recombinant wt or mutant TSEN complexes revealed by phosphorimaging. d Thermal stability of recombinant wt and mutant TSEN complexes assessed by DSF. Shown are means of the denaturation temperature (Td) of each complex with fit errors derived from Boltzmann sigmoids. Fit errors were derived from means of technical triplicates and are representative of biological duplicates (*P = 0.0424, **P = 0.0010, ***P = 0.0004, ****P = 0.0002; unpaired two-tailed Student’s t-test). Source data for a, b, and c, are provided as Source Data files.
Fig. 5
Fig. 5. TSEN54 c.919 G >T fibroblasts exhibit reduced splicing activity in vitro and accumulation of intron-containing pre-tRNAs.
a Pre-tRNA splicing assay (time course) with radioactively labeled S.c. pre-tRNAPheGAA and cell extracts derived from control and PCH patient fibroblasts. Splicing products were separated by urea-PAGE and monitored by phosphorimaging. b Comparison of pre-tRNAIleTAT1-1 intron abundance between control cells and fibroblasts carrying the heterozygous or homozygous TSEN54 c.919 G >T (TSEN54A307S) or the CLP1 c.419 G > A (CLP1R140H) mutation by northern blotting. c Average ratios of pre-tRNAs to mature tRNAs derived from Hydro-tRNAseq for all intron-containing tRNAs comparing PCH patients to wild-type control samples. The black line indicates a slope of 1. d Northern blot analysis comparing pre-tRNAIleTAT1-1 levels to levels of mature tRNAIleTAT or U6 snRNA in control fibroblasts and fibroblasts carrying the heterozygous (het.) or homozygous (hom.) TSEN54 c.919 G > T mutation. The data are representative of three independent experiments. Signal intensities were quantified and displayed as ratios normalized to Ba13 in the bottom panel. Data are presented as mean values ± SD. e Statistical representation of northern blot analysis in d. Mean ratios of signal intensities for pre-tRNAIleTAT1-1 to tRNAIleTAT (left panel) or to U6 snRNA (right panel) were normalized to Ba13, and grouped into control, heterozygous, and homozygous patient classes (n = 3, different control fibroblast cell lines; n = 4, different heterozygous fibroblast cell lines; n = 8, different homozygous PCH patient fibroblast cell lines carrying the TSEN54 c.919 G > T mutation). Unpaired Student´s t-test (two-tailed) for ratios of pre-tRNAIleTAT1-1 to mature tRNAIleTAT or to U6 snRNA revealed a significant difference between control and patient cell lines of 1.2-fold (*P = 0.0371) or 1.3-fold (*P = 0.0344), respectively. Data are presented as mean values ± SD. Panels in a and b are representative of at least two independent experiments. Source data for a, b, c, and e, are provided as Source Data files.
Fig. 6
Fig. 6. Reduced pre-tRNA cleavage activity in PCH patient-derived cell extracts is associated with altered composition of TSEN.
a Comparison of TSEN54 protein levels between control and heterozygous or homozygous PCH patient fibroblasts analyzed by immunoblotting. GAPDH served as a loading control. M, protein size marker. b Co-immunoprecipitation (IP) assay using an α-TSEN34 antibody with cell lysates derived from control and heterozygous or homozygous TSEN54 c.919 G > T fibroblasts analyzed by immunoblotting. The asterisk indicates the heavy chain of the α-TSEN34 antibody. c On-bead pre-tRNA cleavage assay (time course) with radioactively labeled S.c. pre-tRNAPheGAA and immunoprecipitated TSEN complexes (α-TSEN34 antibody-coupled resin) derived from control fibroblasts and from fibroblasts carrying heterozygous or homozygous TSEN54 c.919 G > T mutation shown in (b). Unspecific bands are indicated by asterisks. Data are representative of at least two independent experiments. Source data for a, b, and c, are provided as Source Data files.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schimmel P. The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis. Nat. Rev. Mol. Cell Biol. 2018;19:45–58. doi: 10.1038/nrm.2017.77. - DOI - PubMed
    1. Parisien M, Wang X, Pan T. Diversity of human tRNA genes from the 1000-genomes project. RNA Biol. 2013;10:1853–1867. doi: 10.4161/rna.27361. - DOI - PMC - PubMed
    1. Chan PP, Lowe TM. GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes. Nucleic Acids Res. 2016;44:D184–D189. doi: 10.1093/nar/gkv1309. - DOI - PMC - PubMed
    1. Gogakos T, et al. Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP. Cell Rep. 2017;20:1463–1475. doi: 10.1016/j.celrep.2017.07.029. - DOI - PMC - PubMed
    1. Paushkin SV, Patel M, Furia BS, Peltz SW, Trotta CR. Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3′ end formation. Cell. 2004;117:311–321. doi: 10.1016/S0092-8674(04)00342-3. - DOI - PubMed

Publication types

MeSH terms

Supplementary concepts