Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver - PubMed (original) (raw)

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

C Caudevilla et al. Proc Natl Acad Sci U S A. 1998.

Abstract

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.

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Figures

Figure 1

Figure 1

Three-band pattern obtained by RT-PCR amplification of the mRNAs of rat liver COT. PCR products were obtained by using four sets of primers, E1f-E2r, E1f-E3r, E1f-E5r, and E1f-E7r (A) and E2f-E3r, E2f-E4r, E2f-E5r, and E2f-E7r (B). Schematic representation of the possible origin of PCR products is shown at the top. Exons are indicated by boxes and capital letters; arrows indicate the annealing regions of the PCR primers, lines above denote expected size for the three possible exonic organizations. Representative agarose gels of PCR products obtained with the four sets of primers are shown at the foot of the figure. M indicates the molecular DNA markers. The size and the sequence analysis of PCR products correspond to the exonic representation. The chromatograms show the accurate junction exon 2-exon 2 (C) and exon 3-exon 2 (D).

Figure 2

Figure 2

Southern blot analysis of genomic rat DNA. Total rat genomic DNA, 15 μg per lane, was digested with _Hin_dIII, _Pst_I, _Pvu_II, _Eco_RI, and _Nco_I. Fragments were fractionated electrophoretically, bound on a Hybond-C membrane, and hybridized with a 32P-labeled cDNA probe corresponding to exon 2, with positions 31–166 bp in the cDNA sequence of rat COT (14). DNA molecular size markers are indicated at the right.

Figure 3

Figure 3

Quantification of the RT-PCR products by PCR kinetic analysis cDNAs were obtained from total adult rat liver RNA as described in Materials and Methods. PCR amplifications were performed with E1f and E5r primers (Table 1) in the presence of 2.5 μCi (0.25 μl) of [α-32P]dCTP (300 Ci/mmol). After 20 amplification cycles, a small portion of the PCR mix (10%), was removed every two cycles and the products were resolved on 8% acrylamide gels. The amount of radioactivity in each band was determined by scintillation counting. For kinetic analysis, values of log counts per minute were plotted against the number of cycles. The antilog of the respective intercepts shows the proportion of each mRNA. The upper straight line denotes the 523 pb mRNA (○), and the lower lines correspond to the 659 bp (□) and 784 bp mRNA (▵). The autoradiography is shown in the Inset.

Figure 4

Figure 4

RNase H Digestion of RNA. (A) The diagram shows the products formed after binding the primer E4r to RNA and digesting with RNase H. An analogous diagram can be formulated with the other three primers used. (B) Total RNA from liver of adult rat was bound in different tubes with primers E4r (exon 4), E4r-2 (exon 4), E5r (exon 5), and E7r (exon 7) and then digested with RNase H and analyzed on a 1.6% agarose gel with formaldehyde as described in Materials and Methods (lanes 2–5). Lane 1 denotes control nondigested RNA. The filter containing the transferred RNA was hybridized to a 32P-labeled cDNA probe corresponding to positions 31–1,700 bp in the cDNA sequence from COT (14). RNA molecular size markers (M) are indicated to the right.

Figure 5

Figure 5

Trans-splicing of COT pre-mRNAs in vitro. Transcription plasmids (Bluescript SK+) with the corresponding PCR fragments were obtained as described in Materials and Methods. A Pre-mRNA was 220 nucleotides (nt) long: 55 nt from the plasmid polylinker region (PL) + exon 2 (136 nt) + 25 nt of the 5′ end of intron 2 + 4 nt PL; B Pre-mRNA was 278 long: 23 nt PL + 115 nt of the 3′ end of intron 1 + exon 2 (136 nt) + 4 nt PL. BP indicates branch-point. The 32P-labeled A pre-mRNA was mixed with increasing concentrations of unlabeled B pre-mRNA and incubated in trans-splicing conditions. Lane 2 shows incubation reaction of labeled A pre-mRNA with 50 ng of B pre-mRNA without ATP. Lane 3 shows the incubation reaction with A pre-mRNA but without construct B. A pre-mRNA was incubated with increasing amounts of B pre-mRNA (10 ng, 25 ng, 50 ng, 100 ng, and 200 ng). The trans-splicing product (AxB) (lanes 4–8) was confirmed by RT-PCR and sequencing. The exon 2 band might correspond to the intermediate exon 2 without the attached intron.

Figure 6

Figure 6

Immunolocalization of peroxisomal COT. Ten micrograms of rat liver peroxisomal extracts was separated by SDS/PAGE and subjected to immunoblotting by using specific A43 antibodies (A) for carnitine octanoyltransferase or preimmune sera (B). Two bands corresponding approximately to _M_r of 69 and 79 kDa are observed. The markers (M) were used to determine the approximate molecular weights of the species indicated.

Figure 7

Figure 7

Model for cis- and trans-splicing in COT pre-mRNA. The exons are represented by boxes and the introns by dotted lines. Sequences of the 5′- and 3′-intronic splice sites are indicated in lowercase. The ESE are represented in exon 2 in capital letters. Simultaneous cis- and trans-splicing of hepatic mRNA COT are produced. Trans-splicing can be promoted by ESE sequences. The 3′ acceptor site of intron 1 reacts with the 5′ donor sites of introns 2 or 3 of other molecules of pre-mRNA (trans-splicing). The organization of the mRNAs is shown at the foot.

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References

    1. Bonen L. FASEB J. 1993;7:40–46. - PubMed
    1. Solnick D. Cell. 1985;42:157–164. - PubMed
    1. Konarska M M, Padgett R A, Sharp P A. Cell. 1985;42:165–171. - PubMed
    1. Solnick D. Cell. 1986;44:211. - PubMed
    1. Sharp P A, Konarska M M. Cell. 1986;44:211. - PubMed

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