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.

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA ؉ FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA ؉ FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues. Fibronectins (FN)3 are a family of multifunctional glycoproteins known to play key roles in fundamental processes related to adhesive and migratory behavior of cells, such as embryogenesis, malignancy, homeostasis, wound healing, and maintenance of tissue integrity (1). FN generates protein diversity as a consequence of alternative processing of a single primary transcript at three different sites, the extra domain A (EDA), the extra domain B (EDB), and the type III homologies connecting segment (IIICS) (2-4). Two major forms of FN exist, plasma FN (pFN) and cellular FN. pFN is a soluble dimeric form that is secreted into the bloodstream by hepatocytes (5, 6) and found at 300 and 580 g/ml in plasma of humans and mice, respectively (1, 7). pFN lacks both the EDA and EDB domains, whereas cellular FN is locally produced and deposited as insoluble fibrils in the extracellular matrix of tissues and contains these domains at variable proportions (1, 8, 9). Previous studies suggested that circulating pFN contributes to the extracellular matrix of tissues (10, 11) but the extent of the phenomenon has not been addressed.The levels of FN in plasma are critical for hemostasis, tissue repair, and susceptibility to infections. Depletion of pFN (liver-specific knockout of FN) results in increased brain injury after transient focal cerebral ischemia (12), a delay in thrombus formation and decreased thrombus stability (13), decreased angiogenesis (14), and increased susceptibility to bacterial infections (15). Heterozygous null FN mice appear healthy and fertile (7) but show delayed thrombus growth in injured arterioles (16). Regrettably, the levels of FN present in the tissues of heterozygous null FN and in the pFN null mice have not been reported.We have previously shown that knock-in mice having constitutive inclusion of the EDA exon of the FN gene (EDA ϩ/ϩ strain) had up to 70 -80% reduction ...

In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]).While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.The splicing process is a very flexible and critical step in gene expression. In fact, selected removal or inclusion of individual exons from nascent mRNA molecules allows a single gene to generate multiple proteins with different primary structures; in these cases, the process is known as alternative splicing (1, 38). Constitutive and alternative splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate that has been recently estimated to contain approximately 145 different proteins in addition to the five spliceosomal snRNAs (1,38,41,68). These factors are responsible for accurate positioning of the spliceosome on the 5Ј and 3Ј splice sequences that define the exon. However, correct positioning of the spliceosome is a very complex process owing to the degeneracy of the splice site consensus sequences, the presence of cryptic splice sites in large introns, and the fact that most pre-mRNAs contain multiple introns (26). Therefore, the action of several different proteins is required to achieve accurate positioning of the spliceosome on the splice site. Not surprisingly, alterations in the splicing process have been increasingly reported as being involved in many genetic diseases (5,8,13,23,58). Among the well-known factors that may heavily influence the identification of intron-exon boundaries by the spliceosome are the exon length (3, 65), the presence of splicing enhancer and silencer elements (5, 38), the strength of splicing signals (26), and the promoter architecture (19,33). In addition to these factors, it has been proposed that the natural tendency of RNAs to fold in highly stable secondary and tertia...

Crigler‐Najjar syndrome type I (CNSI) is a rare monogenic disease characterized by severe neonatal unconjugated hyperbilirubinemia with a lifelong risk of neurological damage and death. Liver transplantation is the only curative option, which has several limitations and risks. We applied an in vivo gene targeting approach based on the insertion, without the use of nucleases, of a promoterless therapeutic cDNA into the albumin locus of a mouse model reproducing all major features of CNSI. Neonatal transduction with the donor vector resulted in the complete rescue from neonatal lethality, with a therapeutic reduction in plasma bilirubin lasting for at least 12 months, the latest time point analyzed. Mutant mice, which expressed about 5–6% of WT Ugt1a1 levels, showed normal liver histology and motor‐coordination abilities, suggesting no functional liver or brain abnormalities. These results proved that the promoterless gene therapy is applicable for CNSI, providing therapeutic levels of an intracellular ER membrane‐bound enzyme responsible for a lethal liver metabolic disease.

Alternatively spliced exons generally contain weak splicing sites, and exonic and/or intronic regulatory elements recognised by trans-acting auxiliary splicing factors. The EDA exon of the fibronectin gene is a typical example of an exon bearing a purine-rich exon splicing enhancer (ESE) element recognised by members of the SR phosphoprotein family. The regulatory region that governs splicing in the human EDA exon also contains an exon splicing silencer (ESS) element. We have cloned the mouse EDA genomic region, and we show that the ESE and the ESS elements, although they have base differences, can be replaced by the human elements without significant change in the exon inclusion/exclusion ratio. This fact suggests a common splicing regulatory mechanism across species. We demonstrate in vivo the functional activity of the mouse ESE element in splicing. We also show that the trans-acting factors recognising this element cooperate with the 5P splicing site of the EDA exon to facilitate proper exon recognition. Indeed, a strong 5P splicing site overrides the ESE function in exon recognition. However, the presence of a strong 3P splicing site is not sufficient to compensate for the absence of the splicing enhancer. Our data provide in vivo evidence of the interplay between the exonic splicing regulatory elements and the splicing sites, leading finally to subtle regulation of alternative splicing.z 1998 Federation of European Biochemical Societies.