Acute stress causes expression of a splicing variant of the novel splicing factor (tra2β) and its variant protein in rat gastric mucosa

Abstract

BACKGROUND: The Drosophila transformer-2 (tra2) is a SR-like protein that regulates alternative splicing of pre-mRNAs from several genes with critical roles in sexual differentiation. Recently, two mammalian homologues of the tra2 gene (tra2~nd tra2/]) were molecularly identified. We found that acute stress caused the expression of a splicing variant of the splicing factor (tra2,B) itself in rat gastric mucosa. To understand the physiological meanings of this novel finding, we examined the mechanism for the transcription of the variant, its translation products, cellular and subcellular distribution of the products in gastric mucose. METHODS: All of the tra2/~transcripts hybridized with the rat tra2~ORF cDNA were subcloned and sequenced. Anti-tra2pantibodies were generated by immunization of rabbits with s y ~ c peptides of the amino acid residues 1-20 and 167-183 of rat tra2j~profoln. The levels of tra2/3proteins were measured by Western blotting, Cellular and subcellular iocaiizatbns of tra2pproteins were examined by immunohistochemical analysis. RESULTS: Rat oastdc mucesa constitutively expressed the 1.7and 2.2-kb tra2ptranscripts. Restraint and water-immersion stress selectively decreased the 1.7-kb mRNA expression and inversely induced the expression of a newly transcribed 2.5-kb mRNA. The 17-kb rnRNA lacked e~son 2 that contains muifipla stop codons. Polyadenylation of the transcript produced the 2.2-kb mRNA. The newly transcribed mRNA containd exson 2. From these tra2~nRNAs, two isoforms of tra2/3profeins were translated; 32 kDaand tra2~AN (24 kDa) proteins are synthesized from the ini t i~n codon in exon 1 or the in-frame initiation codon in exon 4, respectively. Rat gestric mucosa constitutively contained these two isoforms. The acute stress rapidly phosphorylated the native 32-kDa protein and increased the tra2/3Z~N level. The phosphorylated tra2~lransiocated into nuclei where spliceosomes were formed, while tra2/3AN lacking phosphorylation sites remained in the cytosol. These subeallular distribution peftems were confirmed by tagged tra2/3Aprotein transtection into AGS cells. CONCLUSION: We demonstrate here that the tra2ppre.mRNA is also subjected to alternative splicing under acute stress, and suggest that acute stress profoundly changes pre-mRNA splicing of distinct genes. The altered RNA-oditing reaction may play an important role in the stress response of gastric mucosa.