Exon repetition: a major pathway for processing mRNA of some genes is allele-specific - PubMed (original) (raw)

Exon repetition: a major pathway for processing mRNA of some genes is allele-specific

Roberto Rigatti et al. Nucleic Acids Res. 2004.

Abstract

Exon repetition describes the presence of tandemly repeated exons in mRNA in the absence of duplications in the genome. Its existence challenges our understanding of gene expression, because the linear organization of sequences in apparently normal genes must be subverted during RNA synthesis or processing. It is restricted to a small number of genes in some of which over half of the mRNA contains specific patterns of repetition. Although it is sometimes assumed to arise by trans-splicing, there is no evidence of this and the efficiency is very much higher than for examples of bona fide trans-splicing in mammals. Furthermore, a potentially ubiquitous reaction such as trans-splicing is not consistent with a phenomenon that involves such a high proportion of the products of so few genes. Instead, it seems more probable that exon repetition is caused by a specific trans-acting factor. We have tested this and demonstrate for the two best characterized examples that the property is restricted to specific alleles of the affected genes and is determined in cis. It is not determined by exonic splicing signals, as had been suggested previously. In heterozygotes, RNA transcribed from the two alleles of an affected gene can have fundamentally different fates.

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Figures

Figure 1

Schematic representation of the mRNA isoforms expressed by the rat COT and Sa genes, and of the primers used in the PCR reactions. (A) Analysis of exon repetition in the COT mRNA. A normal COT mRNA and a COT mRNA in which exon 2 is repeated are illustrated, with (below) an expanded view of exons 1–3 in each case. Primers specific for the COT gene are shown as arrows (5′ to 3′) and named. The COT 2–2 jct primer was designed to amplify a PCR product only if exon 2 is repeated. (B) Analysis of exon repetition in the Sa mRNA. A portion of Sa cDNA is shown (exons 1–6). Three Sa cDNA molecules are depicted. One corresponds to the normal cDNA. The second one contains a repetition of exon 2. Finally, in the third Sa cDNA, exons 2, 3 and 4 are repeated. The primer referred to as SA 2–2 jct specifically amplified transcripts containing a repetition of exon 2, while primer SA 4–2 jct detected the simultaneous repetition of exons 2, 3 and 4.

Figure 2

Exon repetition of COT mRNA in WKY and SHR strains of rat. The presence or absence of exon repetition in COT mRNA was evaluated by RT–PCR followed by separation on agarose gel. Eight WKY and seven SHR animals were used in the experiment. (A) Analysis by amplification with primers COT E1F and COT E7R. This set of primers amplified both the normal COT mRNA and mRNA containing repeated exons, depicted by the adjacent black boxes with white exon numbers. (B) Analysis of COT mRNA by amplification with primers COT E2F and COT E2R, which amplify only a tandem repeat of exon 2.

Figure 3

Segregation of exon repetition in Sa and COT mRNA in an F2 population. Animals from an F2 population derived by crossing F1 individuals (WKY:SHR) were divided into two groups: WKY Sa homozygotes (animals 1–7) and SHR Sa homozygotes (animals 8–16). (A) Analysis of exon repetition in Sa mRNA by RT–PCR with primers SA E1F and SA E2R. The exon organization of the Sa PCR products is shown by the white boxes alongside each PCR product. (B) Analysis of exon repetition in COT mRNA by RT–PCR with primers COT E1F and COT E7R. The exon organization of the COT PCR products is shown by the black boxes. (C) Confirmation of exon repetition in COT mRNA by PCR with primers COT E2F and COT E2R.

Figure 4

Identification of the Sa gene alleles producing mRNA with tandemly repeated exons in heterozygotes. Exon repetition in Sa mRNA was evaluated by RT–PCR in the parental rat strains WKY (W), SHR (S), and in four F2 animals, two of which were homozygous for the WKY Sa allele (animals 1 and 2, the same as in Fig. 2); the other two were heterozygotes, having one copy of the Sa gene from each parent (animals 17 and 18). (A) RT–PCR with primers SA E1F and SA E2R. (B) RT–PCR with primer SA 2–2 jct (which amplifies only Sa isoforms that contain a tandem repeat of exon 2) and primer SA E4R. (C) RT–PCR with primers SA 4–2 jct and SA E4R, to specifically amplify Sa cDNA molecules in which exons 2, 3 and 4 are repeated. (D) An alignment of the first four exons of the WKY and SHR Sa cDNA with the SNP in exon 3 (WKY → G, SHR → A). (E) DNA sequence analysis of Sa exon 3 SNP in PCR products from WKY (W) and SHR (S) alleles mixed in the molar ratios shown. (F) Sequence analysis of RT–PCR products from Sa heterozygotes. PCR products corresponding to the normal Sa mRNA (top), exon 2 repetition (middle) and repetition of exons 2, 3 and 4 (bottom) were produced in separate reactions, purified by agarose gel electrophoresis and analyzed by sequencing. Incorporation of A (SHR allele) is green; G (WKY allele) is black.

Figure 5

Analysis of COT gene alleles involved in exon repetition in heterozygotes. The segregation of the WKY and SHR COT alleles was studied in the F2 population of Figure 3. A SNP in the COT 3′UTR was used to analyze the distribution of the COT alleles in the F2 population. The point mutation creates a recognition sequence for the restriction endonuclease AflII in the WKY COT allele. Thus, amplification products from the COT 3′UTR can be cleaved into two fragments by AflII only if they contain the WKY allele. (A) Identification of COT alleles expressed in total mRNA from F2 animals derived from a WKY × SHR cross. The animals are numbered as in Figure 3. The results are also shown for the parental lines (S, W) and an F1 heterozygote (H). (B) Contributions of the two COT alleles in heterozygotes (animals 7 and 13) to mRNA isoforms. T, COT mRNA was amplified between exons 1 and 17; it was re-amplified by PCR between exons 16 and 17, and analyzed by digestion with AflII and agarose gel electrophoresis. This procedure is similar to that in (A), with prior amplification between exon 1 and 17. ER, exon repetition-containing COT mRNA was amplified between the exon 2–2 jct and exon 17, and then re-amplified and analyzed as for T. (C) Sequence analysis of products of amplification between exons 16 and 17. As in (B), PCR products derived initially by RT–PCR between exons 1 and 17 represent the alleles present in total mRNA, whereas those derived initially by RT–PCR with a primer specific for the tandem repetition of exon 2 represent the alleles present in exon repetition isoforms.

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Exon repetition: a major pathway for processing mRNA of some genes is allele-specific - PubMed (original) (raw)