Loss of seryl-tRNA synthetase (SARS1) causes complex spastic paraplegia and cellular senescence

Abstract

Background: Aminoacyl-tRNA synthetases (ARS) are key enzymes catalysing the first reactions in protein synthesis, with increasingly recognised pleiotropic roles in tumourgenesis, angiogenesis, immune response and lifespan. Germline mutations in several ARS genes have been associated with both recessive and dominant neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase encoded by seryl-tRNA synthetase 1 (SARS1) were reported.

Methods: We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, developmental delay and seizures, but without microcephaly. Complementation and serylation assays using patient's fibroblasts and an Saccharomyces cerevisiae model were performed to examine this variant's pathogenicity.

Results: A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised interleukin-6, p21, p16 and p53 levels).

Conclusion: We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence.

Keywords: genetic research; nervous system diseases; neurology; pediatrics; sequence analysis, RNA.

Figure 1

Seryl-tRNA synthetase 1 (SARS1) de novo variant features. (A) Family tree. Square: male, circle: female, black symbol: affected individual, white symbols: unaffected individuals, WT: wild-type allele. (B) Representation of variant c.969_969+2delGGT impacting on SARS1 splicing. Disruption of exon 7 donor site leads to the use of an alternative AG donor site located 17–18 bases downstream. This results into the inclusion of 16 intronic bp into the coding sequence, and consequently in the in-frame insertion of 5 amino acids. (C) Primary and secondary structure elements of SARS1. Both sequences are shown starting at F(Phe) 316. WT residues: black; inserted residues: blue. Underlined residues correspond to those being part of the conserved motif 2, present in class II aminoacyl-tRNA synthetases. The two residues in red correspond to F321 and E325 that are involved in ATP and serine recognition, respectively. Below each sequence (letters in grey), secondary structure elements are shown (b=β strand, h=α-helix and -=loop region). (D) Three-dimensional model (based on 4L87, modelled with PyMol) showing the ATP/AMP-Ser binding region of SARS1. Cyan: WT model; dark blue: motif 2; red: mutated region (insertion); pink: AMP-serine analogue. (E) Western blot analysis showing SARS1 and γ-tubulin protein levels in fibroblasts from the patient and age-matched controls (n=3). γ-Tubulin was used as a loading control. (F) SARS1 serylation assays in control and patient-derived fibroblast S100 extracts. tRNA concentration was 22 µM. Data were corrected according to the extract’s concentration. Experiments were performed five times independently.

Figure 2

Functional testing using Saccharomyces cerevisiae complementation/serylation assays confirm seryl-tRNA synthetase 1 (SARS1) mutation’s deleterious effects. (A) Growth analysis of S. cerevisiae strains by drop test analysis on SDC-Ura-Leu media. Yeast strain detailed genotypes are detailed in online supplemental table 2. Doubling time of the various strains was determined in liquid SDC-Ura-Leu media in three independent experiments. The results are depicted on the right side. (B) SARS1 serylation assays in S. cerevisiae S100 extracts. tRNA concentration was 22 µM. Data were corrected according to the extract’s concentration. Experiments were performed five times independently.

Figure 3

Patient-derived fibroblasts show senescence-related features. (A) Proliferation measurements. Number of culture days needed for 1 million fibroblasts to reach confluency in 150 mm plates. Patient fibroblasts proliferated at a very much slower rate than controls (n=3). Measures were taken thrice. (B) Immunofluorescence images showing seryl-tRNA synthetase 1 (SARS1) localisation (green) in patient (passage 13) and control fibroblasts (n=4, passages 12–15). Blue: DAPI. Red: gamma-tubulin. Patient fibroblasts’ nuclei are structurally affected and micronuclei can be observed (white arrow). Patient fibroblasts also show cell division abnormalities. (C) Western blot analysis showing H2AX and phosphorylated H2AX (γ-H2AX) protein levels in fibroblasts from the patient (passage 13) and age-matched controls. γ-Tubulin was used as a loading control. (D) β-Galactosidase (β-gal) staining in patient (passage 13) and control fibroblasts (n=4, passages 12–15). Positive β-gal cells (in blue) are highly increased in the patient. Left: representative microscopy images. Right: quantification of β-gal-positive cells (%). (E) qRT-PCR results show upregulation of senescence-associated secretory phenotype (SASP) genes in patient’s fibroblasts compared with controls (n=5). SARS1 and SARS2 genes were also quantified. All experiments were performed three times, independently. The values are represented as the means+SD, and Student’s t-test was performed (*p<0.05; **p<0.01; ***p<0.001).