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Characterization of mouse fibronectin alternative mRNAs reveals an unusual isoform present transiently during liver development.

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.

SUBMITTER: Gorski GK

PROVIDER: S-EPMC6148308 | biostudies-literature | 1996

REPOSITORIES: biostudies-literature

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Characterization of mouse fibronectin alternative mRNAs reveals an unusual isoform present transiently during liver development.

Górski G K GK Aros M C MC Norton P A PA

Gene expression 19960101 3

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 fib ...[more]

PMID: 9041121

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Project description:Alternative splicing of low-voltage-activated T-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. Transcript scanning of the human CACNA1G gene has revealed the presence of 11 regions within the coding sequence subjected to alternative splicing, some of which enhance T-type current. In mouse models of absence epilepsy, elevated T-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. To test whether enhanced T-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse Cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models.Transcript scanning of the murine Cacna1g gene detected 12 regions encoding alternative splice isoforms of Cav3.1/alpha1G T-type calcium channels. Of the 12 splice sites, six displayed homology to the human CACNA1G splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. In addition, two brain region-specific alternative splice patterns were observed in the cerebellum. Comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic T-type currents and wildtype controls failed to reveal differences in Cacna1g splicing patterns.The determination of six novel alternative splice sites within the coding region of the mouse Cacna1g gene greatly expands the potential biophysical diversity of voltage-gated T-type channels in the mouse central nervous system. Although alternative splicing of Cav3.1/alpha1G channels does not explain the enhancement of T-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of T-type channels through this mechanism may influence other developmental neurological phenotypes.

Project description:BACKGROUND:Odorant receptor genes constitute the largest gene family in mammalian genomes and this family has been extensively studied in several species, but to date far less attention has been paid to the characterization of their mRNA 3' untranslated regions (3'UTRs). Given the increasing importance of UTRs in the understanding of RNA metabolism, and the growing interest in alternative polyadenylation especially in the nervous system, we aimed at identifying the alternative isoforms of odorant receptor mRNAs generated through 3'UTR variation. RESULTS:We implemented a dedicated pipeline using IsoSCM instead of Cufflinks to analyze RNA-Seq data from whole olfactory mucosa of adult mice and obtained an extensive description of the 3'UTR isoforms of odorant receptor mRNAs. To validate our bioinformatics approach, we exhaustively analyzed the 3'UTR isoforms produced from 2 pilot genes, using molecular approaches including northern blot and RNA ligation mediated polyadenylation test. Comparison between datasets further validated the pipeline and confirmed the alternative polyadenylation patterns of odorant receptors. Qualitative and quantitative analyses of the annotated 3' regions demonstrate that 1) Odorant receptor 3'UTRs are longer than previously described in the literature; 2) More than 77% of odorant receptor mRNAs are subject to alternative polyadenylation, hence generating at least 2 detectable 3'UTR isoforms; 3) Splicing events in 3'UTRs are restricted to a limited subset of odorant receptor genes; and 4) Comparison between male and female data shows no sex-specific differences in odorant receptor 3'UTR isoforms. CONCLUSIONS:We demonstrated for the first time that odorant receptor genes are extensively subject to alternative polyadenylation. This ground-breaking change to the landscape of 3'UTR isoforms of Olfr mRNAs opens new avenues for investigating their respective functions, especially during the differentiation of olfactory sensory neurons.

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Characterization of mouse fibronectin alternative mRNAs reveals an unusual isoform present transiently during liver development.

Publications

Characterization of mouse fibronectin alternative mRNAs reveals an unusual isoform present transiently during liver development.

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