Experimental evidence for RNA trans-splicing in mammalian cells.

Eul, J.; Graessmann, M.; Graessmann, A.

doi:10.1002/j.1460-2075.1995.tb07325.x

Cited by 67 publications

(89 citation statements)

References 60 publications

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“…The capability of mammalian cells for mRNA trans-splicing was first shown by Eul et al (4). Recently, additional reports demonstrated transsplicing in higher eukaryotes.…”

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confidence: 94%

Transgene analysis proves mRNA trans-splicing at the complex mod(mdg4) locus in Drosophila

Dorn

Reuter

Loewendorf

2001

Proc. Natl. Acad. Sci. U.S.A.

122

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The Drosophila BTB domain containing gene mod(mdg4) produces a large number of protein isoforms combining a common Nterminal region of 402 aa with different C termini. We have deduced the genomic structure of this complex locus and found that at least seven of the mod(mdg4) isoforms are encoded on both of its antiparallel DNA strands, suggesting the generation of mature mRNAs by trans-splicing. In transgenic assays, we demonstrate the ability of Drosophila to produce mod(mdg4) mRNAs by trans-splicing of pre-mRNAs generated from transgenes inserted at distant chromosomal positions. Furthermore, evidence is presented for occurring of trans-splicing of mod(mdg4)-specific exons encoded by the parallel DNA strand. The mod(mdg4) locus represents a new type of comlpex gene structure in which genetic complexity is resolved by extensive trans-splicing, giving important implications for genome sequencing projects. Demonstration of naturally occurring trans-splicing in the model organism Drosophila opens new experimental approaches toward an analysis of the underlying mechanisms.T rans-splicing is a process that produces mature transcripts by combining exons of independent pre-mRNA molecules and was first reported in trypanosomes (1-3). The capability of mammalian cells for mRNA trans-splicing was first shown by Eul et al. (4). Recently, additional reports demonstrated transsplicing in higher eukaryotes. Li et al. (5) identified a splice variant of the human acyltransferase ACAT-1 that contains a 5Ј-untranslated exon encoded from chromosome 7 connected with exons 1-16 encoded from chromosome 1. In rat liver cells, carnitine octanoyltransferase mRNA variants with duplications of coding exons 2 and 3 not present in the genomic complement have been identified (6). In this case, different proteins are encoded by the splice variants. However, the physiological significance of these trans-splicing events remains largely unknown.So far no experimental proof for naturally occurring transsplicing in insects has been documented. Recently, Labrador et al. (7) have shown that the mod(mdg4)-67.2 isoform in Drosophila is encoded on both DNA strands and suggested formation of the mature mRNA by trans-splicing of two independent transcripts.Sequence analysis of a large number of mod(mdg4) cDNA clones (8-12) and the available genomic sequence of the mod-(mdg4) locus (13) indicated that usage of both DNA strands as coding strands is a general property of the mod(mdg4) locus. The common exons 1-4, which are found in all identified transcripts, are located at the 5Ј end of the locus whereas the alternatively spliced 3Ј-specific exons are organized in five groups on both DNA strands. In transgenic assays, we provide evidence that trans-splicing is the basic mechanism responsible for production of multiple mod(mdg4) isoforms. (14) in both orientations. The construct was partially sequenced and used for germline mediated transformation. However, by sequencing the transgene specific reverse transcription (RT)-PCR product, we found a frame shift...

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“…The capability of mammalian cells for mRNA trans-splicing was first shown by Eul et al (4). Recently, additional reports demonstrated transsplicing in higher eukaryotes.…”

mentioning

confidence: 94%

Transgene analysis proves mRNA trans-splicing at the complex mod(mdg4) locus in Drosophila

Dorn

Reuter

Loewendorf

2001

Proc. Natl. Acad. Sci. U.S.A.

122

View full text Add to dashboard Cite

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“…Many external trans-RNAs (i.e. a trans-donor or a trans-acceptor that will participate in a trans-splicing reaction) can be synthesized to interfere with other pre-mRNAs (Bruzik and Maniatis, 1995;Chiara and Reed, 1995;Eul et al, 1995). In the work described here, however, we have chosen a recently discovered natural COT trans-donor and trans-acceptor to study the e ects of interference with the D intron of the H-ras gene.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2001

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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-Hras 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 speci®cally 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 downmodulation 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. Oncogene (2001) 20, 3683 ± 3694.

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“…Furthermore, it has been shown in vitro that Saccharornyces cerevisiae and HeLa cell extracts catalyze the trans-splice reaction [5][6][7][8]. Although predicted by computer analysis [9] and suggested in several earlier reports [5,7,[10][11][12], only recently has conclusive experimental evidence been obtained that intact mammalian cells also have the potential to perform RNA trans-splicing and that the corresponding mRNA is translated into a functional protein [8].…”

Section: Introductionmentioning

confidence: 99%