Biomedical subjects

C Codony

Publications and source records attributed to C Codony.

7 recordsLinked to original sources

PubMed2001

Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.

Alternative Splicing↗

PubMed2001

Modulation in vitro of H-ras oncogene expression by trans-splicing.

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

Animals↗

PubMed2001

Post-transcriptional regulation of rat carnitine octanoyltransferase.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.

Acyl Coenzyme A↗

PubMed1999

Processing of carnitine octanoyltransferase pre-mRNAs by cis and trans-splicing.

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.

Alternative Splicing↗

PubMed1999

Preparation of N2, N2,7-trimethylguanosine affinity columns.

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG-binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.

Animals↗

PubMed1998

Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver.

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.

Animals↗

PubMed1998

A second-step splicing activity is conserved from yeast to human.

We describe a defective HeLa nuclear extract which is particularly deficient in step 2 of splicing reaction. With this extract we have studied the conservation of a second-step activity from yeast to human cells. We detected a S. cerevisiae second-step splicing activity that allows restoration of step 2 of the defective HeLa nuclear extract, which indicates that yeast purified fraction has a second-step activity that is conserved from yeast to human cells. The activity is a yeast UsnRNP protein(s) since it is purified with anti-trimethylguanosine by immunoaffinity columns.

Biological Evolution↗