RESUMO
Carnitine octanoyltransferase (COT), which facilitates the transport of shortened fatty acyl-CoAs from peroxisomes to mitochondria, is expressed in the intestinal mucosa of suckling rats; its mRNA levels increase rapidly after birth, remain steady until day 15, and decrease until weaning, when basal, adult values are established, which remain unchanged thereafter. The process seems to be controlled at the transcriptional level since the developmental pattern of mRNA coincides with that of pre-mRNA values. Dam's milk may influence the intestinal expression of COT, since mRNA levels at birth are low and increase after the first lactation. Moreover, mRNA levels decrease in rats weaned on day 18 or 21. COT is also expressed in the liver of suckling rats. Hepatic COT mRNA is maximal at day 3, remains constant until day 9, and decreases thereafter; this pattern is also similar to that of pre-mRNA values. The profile of expression of COT in intestine and liver strongly resembles that of mitochondrial 3-hydroxy 3-methylglutaryl-coenzyme A synthase and carnitine palmitoyltransferase I, suggesting that analogous transcription factors modulate ketogenesis and mitochondrial and peroxisomal fatty acid oxidation.
Assuntos
Carnitina Aciltransferases/genética , Regulação Enzimológica da Expressão Gênica , Intestinos/enzimologia , Fígado/enzimologia , Animais , Animais Recém-Nascidos , Animais Lactentes , Sequência de Bases , Carnitina Aciltransferases/metabolismo , Éxons/genética , Perfilação da Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Íntrons/genética , Fígado/citologia , Fígado/metabolismo , Dados de Sequência Molecular , Músculo Liso/enzimologia , Músculo Liso/metabolismo , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/genética , DesmameRESUMO
Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process has been described mostly in trypanosoma, nematodes, plant/algal chloroplasts and plant mitochondria [Bonen et al. (1993) FASEB J. 7, 40-46]. Our studies clearly demonstrate that a trans-splicing reaction occurs in the processing of the carnitine octanoyltransferase (COT) gene in rat liver. Three different mature transcripts of COT have been found in vivo, the canonical cis-spliced mRNA and two trans-spliced transcripts, in which either exon 2 or exons 2 and 3 are repeated. Splicing experiments in vitro also indicate the capacity of exon 2 to act either as a donor or as an acceptor of splicing, allowing the trans-splicing reactions to occur.
Assuntos
Processamento Alternativo , Carnitina Aciltransferases/genética , Fígado/enzimologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Éxons , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Peroxissomos/enzimologia , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215-222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5' or the 3' adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected Mr are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.
Assuntos
Carnitina Aciltransferases/genética , Fígado/enzimologia , Precursores de RNA/genética , RNA Mensageiro/genética , Trans-Splicing , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar , Humanos , Dados de Sequência Molecular , RatosRESUMO
The cytosolic enzyme phospho(enol)pyruvate carboxykinase (PEPCK) is markedly expressed in the intestinal mucosa of suckling rats. The expression is located in the small intestine, but there is no expression in stomach, colon, or cecum. The expression changes with age. The mRNA levels at birth are very low, increase after the first lactation, reach maximum levels between 3 and 9 days after birth, and then decrease smoothly. At weaning, when animals begin to feed on a solid chow diet, the expression falls to adult levels, which are hardly detectable. Mother's milk may influence the intestinal expression, since in rats weaned at Day 18, 3 days before normal weaning, the mRNA levels decreased dramatically. mRNA levels for PEPCK in liver present a rather different developmental pattern from that of intestine, remaining high at weaning and in adult rats. On the ninth day after birth, the mRNA levels are the same in intestine and liver.
