A single homozygous point mutation in a 3'untranslated region motif of selenoprotein N mRNA causes SEPN1-related myopathy

Abstract

Mutations in the SEPN1 gene encoding the selenoprotein N (SelN) have been described in different congenital myopathies. Here, we report the first mutation in the selenocysteine insertion sequence (SECIS) of SelN messenger RNA, a hairpin structure located in the 3' untranslated region, in a patient presenting a classical although mild form of rigid spine muscular dystrophy. We detected a significant reduction in both mRNA and protein levels in the patient's skin fibroblasts. The SECIS element is crucial for the insertion of selenocysteine at the reprogrammed UGA codon by recruiting the SECIS-binding protein 2 (SBP2), and we demonstrated that this mutation abolishes SBP2 binding to SECIS in vitro, thereby preventing co-translational incorporation of selenocysteine and SelN synthesis. The identification of this mutation affecting a conserved base in the SECIS functional motif thereby reveals the structural basis for a novel pathological mechanism leading to SEPN1-related myopathy.

Figure 1

Pedigree of the family and SEPN1 mutation in the selenocysteine insertion sequence (SECIS) element. (A) The propositus (II-2) is indicated by an arrow. Affected individuals are indicated by black symbols. No DNA sample was available for individual II-3. Electrophoregrams of nucleotide sequences flanking the mutation in the SECIS element in the 3′ untranslated region of SEPN1. The upper and lower panels represent the control and patient sequences, respectively. The arrow indicates the position of the nucleotide change g.17195T>C (numbering according to SEPN1 genomic sequence from Moghadaszadeh et al, 2001; GenBank accession number NM_020451). (B) Schematic representation of the human selenoprotein N SECIS element showing the hairpin structure composed of two helices (I and II) interrupted by an internal loop and terminated by an apical loop. The position of the mutation affecting the highly conserved non-Watson–Crick base-pair quartet UGAN/NGAN is indicated by an arrow.

Figure 2

Protein levels in cultured skin fibroblasts. Total proteins were extracted from fibroblasts of a control (lane 1) and seven RSMD1 (rigid spine muscular dystrophy) and MmD patients with homozygous SEPN1 mutations (lanes 2–9). Total proteins (50 μg) were resolved on 7% SDS–polyacrylamide gel electrophoresis and analysed by western blotting with an anti-selenoprotein N (SelN) R137 antibody. Lane 1: control; lanes 2 and 9: patient II-2, 50 and 30 μg, respectively; lanes 3 and 4: patients with the c.1386A>G and c.1446delC mutations (Moghadaszadeh et al, 2001); lane 5: c.A1>G mutation affecting the initiation codon; lanes 6 and 7: two patients with c.713–714insA mutation (Ferreiro et al, 2002); lane 8: c.23–32dup10 bp mutation. A monoclonal antibody against α-tubulin was used as a loading control.

Figure 3

Selenoprotein N messenger RNA levels normalized to HPRT (hypoxanthine phosphoribosyltransferase) mRNA. Median values of several quantitative reverse transcription–PCR (n=7 for II-2; n=3 for the patients harbouring the c.1446delC, c.1386A>G and c.A1>G mutations) are presented ±deviation to 25–75%. Statistical significance was determined by a Mann–Whitney rank sum test (^*P=0.001; ^**P=0.024).

Figure 4

The binding activity of selenocysteine insertion sequence (SECIS)-binding protein 2 (SBP2) to the SECIS element harbouring the patient mutation. A DNA fragment containing the selenoprotein N SECIS element (corresponding to position 17187–17245 of genomic DNA) was PCR amplified and cloned downstream of a T7 RNA polymerase transcription start site. The U to C mutation was introduced by site-directed mutagenesis. Both wild-type and mutated SECIS RNAs were produced and radiolabelled by T7 transcription in the presence of [α-³²P]ATP. Either wild-type SECIS RNA (lanes 1–5) or mutated (lanes 6–10) ³²P-labelled SECIS RNA was incubated in the presence of increasing amounts of recombinant SBP2 protein (200 ng, lanes 2 and 7; 400 ng, lanes 3 and 8; 800 ng, lanes 4 and 9; 1,000 ng, lanes 5 and 10). The protein/RNA complexes were resolved on a 6% non-denaturing polyacrylamide gel and visualized by autoradiography. Migration was compared with the signal obtained in the absence of SBP2 (lanes 1 and 6). Migration positions of the free SECIS RNA and SBP2/SECIS complexes are shown.