CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration.

Identification of a homozygous CLP1 p.R140H mutation in families with degeneration/hypoplasia of the central nervous system. Further analysis in Figure S1. (A) Pedigrees of four consanguineous Turkish families. Filled symbols: affecteds; hash: deceased; double bar: consanguinity; dashed double bar: history of consanguinity but ancestry not established. (B) Midline sagittal (top) and coronal (bottom) brain MRI of control compared with patients from each family, showing ventriculomegaly due to atrophy. Red arrowhead: hypoplastic/atrophic pons. White arrowhead: cerebellar folia atrophy. Double white arrowheads: hypoplastic corpus callosum. Red asterisk: Fluid cavity as a result of cerebellar atrophy (mega cisterna magna). Only axial CT was available for 1337-II-1. (C) Stick figure of CLP1 protein and location of the p.R140H mutation near the ATP-binding P-loop. Evolutionary conservation of the p.R140 residue across the animal kingdom. NTD: N-terminal domain; P-loop/Walker A motif; Switch loop I; Switch loop II; base-binding loop: involved in nucleotide binding; CTD: C-terminal domain. H.s.: Human; M.m.: Mouse; G.g.: Chicken; X.l.: Frog; D.m.: Fly; C.e.: Worm; S.c.: Yeast.

CLP1 p.R140H is stable but functionally compromised. (A) Homology modeling of human CLP1 with the crystal structure of yeast Clp1 (left). Lower panels: Magnified images of the p.140R and p.140H residues in human and p.149K residue in yeast. Substitution predicted to disrupt conserved hydrogen bond (red arrowhead) formed between p.R140 and GLU (red arrow, residue 48 in human and 59 in yeast). (B) Unaltered levels of CLP1 protein from affected cells. (C) Defective kinase activity of recombinant human CLP1 p.R140H mutation (purification shown in Figure S2), against RNA poly(A)20, RNA SYBR staining (bottom), quantified at right. (D) Nuclear localization of CLP1 p.R140H is reduced in affected cells (schematic and validation shown in Figure S2). (E) Western blot of cellular fractions showing CLP1 mislocalization in patient fibroblasts. TFIIH, β-ACTIN, and GAPDH: loading controls, quantified below. N = nuclear, C= cytoplasmic, Unaff. = Unaffected, Aff. = Affected. Error bar: SEM. * p < 0.05 Student's t-test. Scale bar = 10 μm.

Zebrafish clp1R44X homozygous mutant show gross brain defects, reduced survival and neurodegeneration. (A) Gross morphology of wt and clp1R44X/R44X zebrafish mutant showed misshapen head, small eye and curved tail (arrow), suggesting neuromotor defects. Scale bar = 500 μm (B) Kaplan–Meier curve showed reduced survival of clp1R44X/R44X fish (additional allele shown in Figure S3). (C) Progressively reduced otx2 expression in developing clp1R44X/R44X zebrafish brains. Broad otx2 expression domain at 24 hpf was unremarkable in mutant (bracket), suggesting initial patterning was not disrupted. From 48-72 hpf, wt fish showed expression restricted to midbrain-hindbrain organizer (bracket), whereas mutant showed weak expression, completely absent by 72 hpf (#). (D) TUNEL positive cells were dramatically increased in mutant at 48 hpf in both the hindbrain (arrow) and the midbrain/diencephalon (double arrow), further investigated in Figure S3. (E) Partial rescue of the clp1R44X/R44X phenotype with human wt but not p.R140H CLP1 mRNA, by measuring curved tail height. No significance difference was detected between uninjected and injected with p.R140H mRNA, whereas wt mRNA partially recovered curved tail phenotype. p <0.01 student t-test. (F) In situ for otx2 in wt, clp1R44X/R44X, and clp1R44X/R44X injected with human CLP1 mRNA. Human CLP1, but not CLP1R140H, prevented the loss of otx2 expression at 48 hpf in clp1 mutants, quantified at right. n>25 embryos each condition, p < 0.05 Student's t-test. Scale bar = 100 μm.

Increased intron-containing pre-tRNA in CLP1 mutant patient cells and loss of TSEN complex affinity resulting in reduced pre-tRNA cleavage with CLP1-purified complexes. (A) Schematic pre-tRNA with location of the intron, occurring one base 3’ to the anticodon. Primers designed to the individual tRNA homologue exon sequence and the unique consensus intron (red). Amplifications with the 5’-primer and any of the 3’-primers. (B, C) qRT-PCR results showed variable changes in pre-tRNA expression in Affected fibroblasts (i.e. about half were increased and half were decreased in Affected), while most pre-tRNA transcripts from Affected iNeurons were increased (i.e. six of eight were increased). Validation and unchanged pre-tRNAs are shown in Figure S4. (D) Northern blot analysis of Chr14:tRNA19-TyrGTA and Chr19:tRNA10-IleTAT pre-tRNA (intron probes) and tRNA-TyrGTA, tRNA-IleTAT, tRNA-LeuCAA pan mature tRNA (5’-exon probes) transcripts relative to U6 loading control, in duplicate showed similar amounts of pre-tRNA and mature tRNA for all transcripts tested, while Affected iNeurons show a increase in Tyr, Ile, and Leu pre-tRNA transcripts, with a corresponding reduction in processed, mature tRNA. Quantification below, displayed as percent mature or pre-tRNA of total tRNA. (E) Western blot of CLP1-purified complexes showing CLP1 p.R140H, p.R140A, and p.K127A with reduced bound TSEN2 and TSEN34 compared to wt CLP1 (TSEN54 served as a positive control). Quantification of the amount of TSEN2 normalized to FlagHis-tagged protein from four independent replicates (below). (F) Time-course of tRNA endonuclease reactions performed on exogenous radiolabeled Tyrosine (Tyr) pre-tRNA with double affinity purified CLP1-bound complexes. Buffer serves as a negative control and Yeast tRNA endonuclease as a positive control. Reduced endonuclease activity observed with CLP1R140H bound complexes compared with wt, quantified at left. *= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001 Students t-test (qPCRs) or one-way ANOVA (pulldown).

Toxic effects of unprocessed 3’-“half” tRNA exon in CLP1 patient cells. (A) Model of tRNA splicing. The pre-tRNA contains an intron (red) 1 base 3’ of the anticodon. The TSEN complex excises the intron, leaving two “half” tRNAs, the 5’-exon containing a 2’-3’-phosphodiester, and the 3’-exon containing an hydroxyl group. CLP1 is capable of phosphorylating the 5’-end of the 3’-exon, then a still unknown ligase repairs the break. (B, C) Control (green) and CLP1 patient cells (red) transfected with either the unphosphorylated 3’-exon (3’-Exon), or the phosphorylated 3’-exon (P-3’-Exon), in the presence or absence of hydrogen peroxide. None of the conditions were adverse to control cells (shown in Figure S5), whereas patient cells showed reduced viability to hydrogen peroxide and 3’-Exon transfection, and improved viability upon P-3’-Exon transfection. * p < 0.05 2-way ANOVA.