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Characterization of mouse fibronectin alternative mRNAs reveals an unusual isoform present transiently during liver
G K Górski1, M C Aros, P A Norton
1Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107, USA.
Abstract:
Fibronectins are found in many extracellular matrices as well as being abundant plasma proteins. The plasma isoforms of fibronectin, which are synthesized in the adult by liver hepatocytes, differ from those derived from most other cells and tissues due to alternative mRNA splicing. Studies in several vertebrates have indicated that FN alternative splicing is regulated spatially and temporally during development. The mouse represents an attractive organism in which to study the regulation of fibronectin splicing during development, but the patterns of fibronectin alternative splicing were not known for this species. Mouse fibronectin cDNA clones were isolated and sequenced, revealing > 95% identity with rat fibronectin at the amino acid level; all three segments that undergo alternative splicing are well conserved. RNase protection and RT-PCR were used to determine the patterns of alternative splicing that occur in fibroblasts and adult liver, sources of cellular and plasma fibronectins. Only A-B-mRNAs were detected in liver, and three V region variants were observed, corresponding to the protein isoforms V120, V95, and V0. Fibroblasts produced mRNAs that were heterogeneous for A and B splicing, but all RNAs contained V120. These patterns contrast with the embryonic form (B+A+V120). Characterization of fibronectin mRNAs from livers of fetal and newborn mice revealed that a significant level of B+ mRNA was present throughout late gestation, declining at birth. Little A+ mRNA was present, and the adult liver V region pattern was observed at all stages. Thus, fibronectin splicing changes during liver development are noncoordinate. One consequence of this temporal regulation is the transient synthesis of B+ mRNAs, including a novel isoform, B+A-V0.
Insights
Fibronectin alternative splicing in mice differs between liver and fibroblasts. Liver fibronectin splicing patterns change non-coordinately during development, leading to transient novel isoforms.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Fibronectins (FN) are extracellular matrix and plasma proteins crucial for development.
- FN alternative mRNA splicing generates diverse protein isoforms, with patterns varying by cell type and developmental stage.
- Understanding FN splicing regulation in mice is vital, but patterns were previously unknown for this species.
Purpose of the Study:
- To characterize fibronectin (FN) alternative mRNA splicing patterns in mouse fibroblasts and adult liver.
- To investigate the temporal regulation of FN splicing during mouse liver development.
- To identify novel FN isoforms generated through developmental splicing changes.
Main Methods:
- Isolation and sequencing of mouse fibronectin cDNA clones.
- RNase protection assays and RT-PCR to analyze mRNA splicing patterns.
- Comparison of splicing patterns in fetal, newborn, and adult mouse liver, as well as fibroblasts.
Main Results:
- Mouse FN cDNA showed high amino acid identity (>95%) with rat FN, with conserved alternatively spliced segments.
- Adult mouse liver primarily produced A-B-mRNAs with V120, V95, and V0 variants; fibroblasts produced heterogeneous A/B splicing but consistently V120.
- Fetal and newborn mouse livers exhibited significant B+ mRNA, declining at birth, while adult liver patterns were established early; A+ mRNA was minimal.
- A novel B+A-V0 isoform was transiently synthesized during late gestation.
Conclusions:
- Fibronectin splicing in mouse liver and fibroblasts exhibits distinct patterns.
- Liver fibronectin splicing undergoes non-coordinate temporal regulation during development.
- Developmental changes in mouse liver FN splicing result in the transient production of specific isoforms, including a novel B+A-V0 variant.