RESUMO
Gas7, a growth arrest-specific gene first isolated from serum-starved NIH3T3 cells, is expressed abundantly in the brain and is essential for the outgrowth of neurites from cultured cerebellar neurons. Here, we report the existence of a Gas7-related cDNA, designated Gas7-cb, isolated from the mouse cerebellum, and we report the finding that Gas7-cb transcripts and protein are expressed at different locations than those of Gas7. Gas7-cb cDNA differs from the Gas7 cDNA only in the 5' region. Its encoded protein shares the same 320 amino acids in its C-terminus with those of Gas7. Analyses of the RNA and protein expression of Gas7-cb and Gas7 by RNase protection assay and Western blot indicated that while Gas7 expression is predominant in the cerebrum and in growth-arrested NIH3T3 fibroblasts, Gas7-cb expression is predominant in the cerebellum. Characterization of Gas7 and Gas7-cb RNAs and of the genomic structure of murine Gas7 cloned in a bacterial artificial chromosome indicated that the Gas7 gene spans more than 60 kb and consists of at least 15 exons. The 5'-terminus of Gas7-cb is located at exon 6a, which is absent in Gas7 transcripts but is retained in its entirety in Gas7-cb transcripts, resulting in the presence of a unique 20-amino-acid sequence at the N-terminus of the Gas7-cb protein. Our results show that the Gas7 gene encodes two Gas7 isoforms, Gas7 and Gas7-cb, whose expression is differentially regulated within mouse brain.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células 3T3 , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Southern Blotting , Western Blotting , Mapeamento Cromossômico , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons/genética , Perfilação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonucleases/metabolismo , Análise de Sequência de DNARESUMO
RNase E is encoded by the rne (also known as ams or hmp) gene and is the principal enzyme that controls the chemical decay of bulk mRNA in Escherichia coli. Earlier work has shown that RNase E degrades its own mRNA, autoregulating production of the RNase E protein. Here we show that in cells lacking RNase E activity, the 3.6-kilobase rne gene transcript is cleaved site specifically at two locations near its center by a novel endonuclease whose activity is modulated by the presence or absence of amino acids in the culture medium. These cleavages produce a 2-kilobase RNase E-sensitive RNA fragment corresponding to the 3' half of the transcript. Using primer extension and RNase protection analysis, we mapped RNase E-independent cleavages to sites 1558 and 1576 nucleotides from the 5' end of the rne transcript (coordinates 1738 and 1747 of the rne gene). Our results indicate the existence of a previously unknown RNase E-independent mechanism for degradation of rne transcripts and further demonstrate that this mechanism responds to changes in cell growth conditions.
