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Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

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Related in: MedlinePlus

Regulated splicing of the EDA is dispensable for the ECM formation. (A) RT-PCR analysis of total RNA prepared from MEF from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d postcoitus [p.c.]). (B and C) Western blot analysis of protein extracts prepared from the above-described MEF (20 and 100 μg for B and C, respectively) with anti-FN and anti-EDA antibodies, respectively. Coomassie blue staining showed equal loading. (D) MEF prepared from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d p.c.) were plated, fixed, and incubated with anti-EDA antibody (left column) or with anti–total FN antibody (middle column). The merged image is shown in the right column.
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fig2: Regulated splicing of the EDA is dispensable for the ECM formation. (A) RT-PCR analysis of total RNA prepared from MEF from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d postcoitus [p.c.]). (B and C) Western blot analysis of protein extracts prepared from the above-described MEF (20 and 100 μg for B and C, respectively) with anti-FN and anti-EDA antibodies, respectively. Coomassie blue staining showed equal loading. (D) MEF prepared from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d p.c.) were plated, fixed, and incubated with anti-EDA antibody (left column) or with anti–total FN antibody (middle column). The merged image is shown in the right column.

Mentions: Next, we examined the expression of FN mRNA, protein isoforms and ECM formation in MEF prepared from EDAwt/wt, EDA+/+, and EDA−/− animals. RT-PCR analysis confirmed the constitutive splicing-in of the EDA exon in the EDA+/+ and its absence in the EDA−/− MEFs (Fig. 2Figure 2.


Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

Regulated splicing of the EDA is dispensable for the ECM formation. (A) RT-PCR analysis of total RNA prepared from MEF from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d postcoitus [p.c.]). (B and C) Western blot analysis of protein extracts prepared from the above-described MEF (20 and 100 μg for B and C, respectively) with anti-FN and anti-EDA antibodies, respectively. Coomassie blue staining showed equal loading. (D) MEF prepared from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d p.c.) were plated, fixed, and incubated with anti-EDA antibody (left column) or with anti–total FN antibody (middle column). The merged image is shown in the right column.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172721&req=5

fig2: Regulated splicing of the EDA is dispensable for the ECM formation. (A) RT-PCR analysis of total RNA prepared from MEF from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d postcoitus [p.c.]). (B and C) Western blot analysis of protein extracts prepared from the above-described MEF (20 and 100 μg for B and C, respectively) with anti-FN and anti-EDA antibodies, respectively. Coomassie blue staining showed equal loading. (D) MEF prepared from EDAwt/wt, EDA+/+, and EDA−/− embryos (13.5 d p.c.) were plated, fixed, and incubated with anti-EDA antibody (left column) or with anti–total FN antibody (middle column). The merged image is shown in the right column.
Mentions: Next, we examined the expression of FN mRNA, protein isoforms and ECM formation in MEF prepared from EDAwt/wt, EDA+/+, and EDA−/− animals. RT-PCR analysis confirmed the constitutive splicing-in of the EDA exon in the EDA+/+ and its absence in the EDA−/− MEFs (Fig. 2Figure 2.

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

Show MeSH
Related in: MedlinePlus