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The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression
J V Barnier1, C Papin, A Eychène
1Unité Mixte de Recherche 146 du CNRS, Institut Curie, Centre Universitaire, Orsay, France.
Abstract:
The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.
Insights
Researchers discovered distinct B-Raf protein isoforms in mouse tissues, generated by alternative splicing of exons 8b and 10. These B-Raf variants, particularly those with exon 10, show tissue-specific expression, suggesting specialized roles in cellular signaling pathways.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Genetics
Background:
- The c-Rmil/B-raf proto-oncogene encodes serine/threonine protein kinases crucial for signal transduction.
- B-raf is implicated in transmitting signals from the cell membrane to the nucleus.
Purpose of the Study:
- To investigate the alternatively spliced exons of B-raf and identify resulting protein isoforms.
- To determine the tissue-specific expression patterns of B-Raf proteins in adult mice.
Main Methods:
- Isolation of B-raf cDNAs from a mouse brain library.
- Identification of protein isoforms using specific antibodies against B-Raf.
- Analysis of B-raf mRNA expression via reverse transcriptase-polymerase chain reaction (RT-PCR).
Main Results:
- Discovery of a novel alternatively spliced exon (exon 8b) in mouse B-raf.
- Identification of at least 10 distinct B-Raf protein isoforms in adult mouse tissues.
- Demonstration of tissue-specific expression of B-Raf isoforms, with exon 10-containing variants found predominantly in neural tissues.
Conclusions:
- Alternative splicing of B-raf exons 8b and 10 generates multiple protein isoforms.
- Distinct B-Raf proteins exhibit tissue-specific expression patterns.
- These findings suggest specialized roles for different B-Raf isoforms in tissue-specific signal transduction pathways.