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

Kazuhisa Yamazaki,Kei Arimatsu,Takako Ida,Sayuri Miyauchi,Hiromasa Yoshie,Tomoki Maekawa,Kasper Hoebe,Kinji Ohno,Bruce Beutler,Yumi Matsuda,Mai Yokoji,Naoki Takahashi,Karine Crozat,Tomoka Hasegawa,Eva Marie Y. Moresco,Norio Amizuka,Masaru Kaku,Xin Du,Koichi Tabeta

doi:10.1038/s41598-017-10343-9

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. Col1a1seal 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 Col1a1seal 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.”

Highlights

GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes
Splice donor and acceptor site mutations can lead to exon skipping, use of cryptic splice sites, and/or insertions/deletions; many human diseases stem from such defects caused by splice site mutations, and their mechanisms have been well documented[12]
Because a glycine residue at every third position of the chain is critical to the formation of the triple helix of mature type I collagen[21], frameshifts or premature termination caused by aberrant splicing can be detrimental for type I collagen synthesis[15,16]

Summary

Introduction

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. COL1A1 and COL1A2 contain approximately 52 intronic sequences that need to be precisely excised to generate mature mRNA, making these genes susceptible to splicing mutations[7,8]. Greater than 99% of mammalian introns, including all of those in COL1A1, are spliced by the major (U2-dependent) spliceosome and have as their terminal dinucleotides GU (5′ end) and AG (3′ end)[10]. Exon skipping mutations causing lethal or moderate phenotypes of OI have been identified in both splice donor and acceptor consensus sequences in COL1A1 and COL1A2 genes[15,16]. Novel causative mutations for OI continue to be identified[17]

Methods

Results

Conclusion