An ENU-induced splice site mutation of mouse Col1a1 causing recessive osteogenesis imperfecta and revealing a novel splicing rescue

Abstract

GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T → A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, α1 chain, was responsible for the phenotype. Col1a1 ^seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1 ^seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing "rescue."

Figure 1

Phenotype of seal mutant mice. All the specimens were from male mice. (A) Wild-type and seal homozygous mice (12 weeks of age). (B) Femur and tibia of a 12-week-old seal homozygous mouse. They are abnormal in shape and shorter than the corresponding wild-type bones. Bone marrow is visible through thin cortical bone of seal homozygous animals. Scale: length of smallest grid square, 5 mm. (C) Femur length. N = 5 mice per genotype. (D) Swollen and deformed foot of a seal homozygote. (E) Body weight of age-matched mice. N = 5 mice per genotype. In C and E, results are expressed as mean ± SD.

Figure 2

Genetic mapping and identification of the seal mutation. (A) The seal mutation was mapped to chromosome 11 on 34 meioses with peak LOD score of 6.9. Phenotypic classification was based on the inducible defect in hind limb movement. (B) Fine mapping of the mutation on chromosome 11 using microsatellite markers. The seal mutation was confined to a critical region marked by D11mit289 and D11mit67 with five crossover events proximal and three crossovers distal to the mutation. (C) DNA sequence chromatograms of the region containing the seal mutation. (D) Illustration of the position of the seal mutation relative to exons 36 and 37 of Col1a1.

Figure 3

Abnormal bone structure and collagen network in seal homozygotes. Light microscopic (A–D) or TEM imaging (E–J) of the tibial sections from wild-type and seal homozygous mice. (A, B) The proximal region of the tibia. (C, D) Enlargement of the regions boxed in A and B, the metaphysis. Many thin trabeculae (arrowheads in D) were found in seal metaphysis (meta) compared with those (arrowheads in C) in wild-type mice. (E, F) Osteoblasts (ob) of the tibia. (G, H) Enlargement of the regions boxed in E and F, the bone matrix. Wild-type bone matrix demonstrated dense collagen fibrils (black fibrillar structures), whereas seal bone matrix contained sparsely-distributed collagen fibrils featuring organic materials (arrows). (I, J) Both wild-type and seal homozygous osteoblasts in the metaphyses of tibiae were cuboidal in shape, and showed developed rough endoplasmic reticulum (rER) and Golgi apparatus (Go). Scale bars, A, B: 500 μm, C, D: 100 μm, E, F: 2 μm, G-J: 0.5 μm.

Figure 4

Thin cortical bone in seal femurs. Representative micro-CT images for wild-type mice (left) and seal homozygotes (right). (A) Sagittal sections. (B) Cross section of mid-diaphysis.

Figure 5

Metabolic balance of type I collagen in bone. CTX level in serum. N = 3 mice per genotype.

Figure 6

Collagen quantification and component assay. (A) Hydroxyproline content in demineralized bone hydrolysate was measured as an indicator of collagen content. (B) Representative gel image of femur type I collagen components separated by SDS-PAGE and visualized by CBB. For β-chains, numbers following the β designation indicate the identity of the two α-chain components [e.g. β12 is a heterodimer of α1 (I) and α2 (I)]. Band intensity represents collagen extractability. (C) Gel bands from B were quantitated by densitometric image analysis. Sum of quantitated band intensities of all type I collagen chains, representing collagen extractability, was plotted (normalized to wild-type). (D) Ratio of band intensities of α1 (I) and α2 (I) chains (α1 (I)/α2 (I) chain ratio). (E) Quantitation of β-chains by densitometric image analysis of CBB-stained SDS-PAGE gel containing type I collagen components from an independent extraction from femur samples. N = 3 mice per genotype. Data are expressed as mean ± SEM.

Figure 7

Reduced type I collagen gene expression in seal mice. Col1a1 gene expression relative to Gapdh in the femur were measured by quantitative RT-PCR using primer sets targeting sequences in exons 36 and 37, or exons 40 and 41. The ratio of Col1a1 exon 36–37/40–41 was analyzed for total RNA (A), nuclear RNA (B), and cytoplasmic RNA (C). P-values, (A) P = 0.007, P = 0.002, and n.s. from left, (B) P = 0.036, P = 0.040, and n.s. from left, and (C) P = 0.002, P = 0.027, and n.s. from left. N = 3 mice per genotype. Data are expressed as mean ± SEM.

Figure 8

Minigene assay for Col1a1 ^seal pre-mRNA splicing. (A) DNA sequence chromatograms of femur RT-PCR amplification products generated using primers complementary to sequences in exons 26 and 39. (B) Schematic illustration of Col1a1 ^seal exons used in minigenes exon 25–45, 26–39 + intron 25, 26–39, and 34–43. Red and blue coloring denotes minigenes spliced without or with exon 36, respectively. (C) Sequence analyses and gel images of spliced transcripts of Col1a1 ^seal minigene exon 26–39 (right). The major splice product from Col1a1 ^seal minigene exon 26–39 lacked exon 36 and gel image showed corresponding band was shifted down compared with wild-type minigene band. In contrast, the majority of transcripts from Col1a1 ^seal minigene exon 26–39 + intron 25 (left) were correctly spliced, with a minor fraction having exon 36 completely skipped which was not observed in vivo.