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B-raf alternative splicing is dispensable for development but required for learning and memory associated with the hippocampus in the adult mouse.

Valluet A, Hmitou I, Davis S, Druillennec S, Larcher M, Laroche S, Eychène A - PLoS ONE (2010)

Bottom Line: It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory.However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory.Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Orsay, France.

ABSTRACT
The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.

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Phylogenetic analysis of B-raf exon 8b.(A) Schematic representation of B-raf genomic structure between exon 8 and exon 9 in different species. Positioning of intronic and exonic sequences was obtained using ClustalW2 multiple sequence alignment program as described in Suplementary Figure. The size of introns is indicated. Exon 8b sequences are found only in eutherians, but not in other mammals and in chicken. (B) Exon 8b amino-acid sequence conservation in eutherians. Evidence for the presence of B-raf exon 8b sequences on genomic DNA (G) or transcripts (T) is indicated on the right. B-raf transcripts containing exon 8b have been reported in mouse (This study and Barnier et al., 1995 [17]; cDNA accession number AJ276308) and rat (EST accession number BF543389). (C) Exon 8b is not expressed in human brain. B-Raf isoforms expression was analyzed in human and mouse total brain by RT-PCR as described in Materials and Methods.
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pone-0015272-g001: Phylogenetic analysis of B-raf exon 8b.(A) Schematic representation of B-raf genomic structure between exon 8 and exon 9 in different species. Positioning of intronic and exonic sequences was obtained using ClustalW2 multiple sequence alignment program as described in Suplementary Figure. The size of introns is indicated. Exon 8b sequences are found only in eutherians, but not in other mammals and in chicken. (B) Exon 8b amino-acid sequence conservation in eutherians. Evidence for the presence of B-raf exon 8b sequences on genomic DNA (G) or transcripts (T) is indicated on the right. B-raf transcripts containing exon 8b have been reported in mouse (This study and Barnier et al., 1995 [17]; cDNA accession number AJ276308) and rat (EST accession number BF543389). (C) Exon 8b is not expressed in human brain. B-Raf isoforms expression was analyzed in human and mouse total brain by RT-PCR as described in Materials and Methods.

Mentions: We previously reported the high conservation of B-raf exon 9b in vertebrates and its presence in transcripts from a wide variety of species, including fish, amphibians, avians and mammals [19]. In contrast, scarce information was available concerning exon 8b. For example, exon 8b-containing B-raf transcripts have been reported only in mouse and rat, thus far (Figure 1B and C). A search for B-raf exon 8b sequences in available genomic libraries revealed the presence of exon 8b only in eutherians, but not in other mammals such as opossum (marsupialia) and platypus (monotremata), or in avian species (Figure 1A and Figure S1). Despite the presence of exon 8b on genomic DNA from primates, we failed to detect exon 8b-containing transcripts in human brain, testis, heart and various cell lines (Figure 1C and data not shown).


B-raf alternative splicing is dispensable for development but required for learning and memory associated with the hippocampus in the adult mouse.

Valluet A, Hmitou I, Davis S, Druillennec S, Larcher M, Laroche S, Eychène A - PLoS ONE (2010)

Phylogenetic analysis of B-raf exon 8b.(A) Schematic representation of B-raf genomic structure between exon 8 and exon 9 in different species. Positioning of intronic and exonic sequences was obtained using ClustalW2 multiple sequence alignment program as described in Suplementary Figure. The size of introns is indicated. Exon 8b sequences are found only in eutherians, but not in other mammals and in chicken. (B) Exon 8b amino-acid sequence conservation in eutherians. Evidence for the presence of B-raf exon 8b sequences on genomic DNA (G) or transcripts (T) is indicated on the right. B-raf transcripts containing exon 8b have been reported in mouse (This study and Barnier et al., 1995 [17]; cDNA accession number AJ276308) and rat (EST accession number BF543389). (C) Exon 8b is not expressed in human brain. B-Raf isoforms expression was analyzed in human and mouse total brain by RT-PCR as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008692&req=5

pone-0015272-g001: Phylogenetic analysis of B-raf exon 8b.(A) Schematic representation of B-raf genomic structure between exon 8 and exon 9 in different species. Positioning of intronic and exonic sequences was obtained using ClustalW2 multiple sequence alignment program as described in Suplementary Figure. The size of introns is indicated. Exon 8b sequences are found only in eutherians, but not in other mammals and in chicken. (B) Exon 8b amino-acid sequence conservation in eutherians. Evidence for the presence of B-raf exon 8b sequences on genomic DNA (G) or transcripts (T) is indicated on the right. B-raf transcripts containing exon 8b have been reported in mouse (This study and Barnier et al., 1995 [17]; cDNA accession number AJ276308) and rat (EST accession number BF543389). (C) Exon 8b is not expressed in human brain. B-Raf isoforms expression was analyzed in human and mouse total brain by RT-PCR as described in Materials and Methods.
Mentions: We previously reported the high conservation of B-raf exon 9b in vertebrates and its presence in transcripts from a wide variety of species, including fish, amphibians, avians and mammals [19]. In contrast, scarce information was available concerning exon 8b. For example, exon 8b-containing B-raf transcripts have been reported only in mouse and rat, thus far (Figure 1B and C). A search for B-raf exon 8b sequences in available genomic libraries revealed the presence of exon 8b only in eutherians, but not in other mammals such as opossum (marsupialia) and platypus (monotremata), or in avian species (Figure 1A and Figure S1). Despite the presence of exon 8b on genomic DNA from primates, we failed to detect exon 8b-containing transcripts in human brain, testis, heart and various cell lines (Figure 1C and data not shown).

Bottom Line: It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory.However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory.Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Orsay, France.

ABSTRACT
The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.

Show MeSH
Related in: MedlinePlus