Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites

Abstract

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.

Figure 1

DNA target sites and COXI (cytochrome c oxidase subunit I) gene rearrangements caused by transposition of aI1. (A) DNA target-site sequences. The sequence of the aI1 homing site is shown from position −23 in exon 1 (E1) to +10 in exon 2 (E2). The IBS1, IBS2, and δ′ sequences are indicated, with the complementary intron RNA sequences EBS1, EBS2, and δ shown above. The base-pairing interaction at position −6 of EBS1/IBS1, which is disrupted by the A262T mutation in the intron RNA, is boxed. The sense-strand sequences of the E1/aI1 and 5β848 ectopic sites are shown below. Asterisks indicate nucleotide residues that are conserved between the natural site and the ectopic sites. DNA and RNA sequences are indicated in uppercase and lowercase letters, respectively. (B) Diagram of the COXI gene and products of aI1 transposition in strain 1^+t2⁰. Exons are filled rectangles and intron ORFs are hatched (aI1) or open (aI3α–aI5β) rectangles. Five ectopic sites for aI1 insertion are indicated below with bold arrows indicating the sites analyzed here. (i) aI1 insertion at the 5β848 site. (ii) COXI deletion resulting from aI1 insertion at the 5β848 ectopic site, followed by recombination between the two copies of aI1. (iii) aI1 insertion at the E1/aI1 site resulting in two tandem copies of aI1 (“intron dimer”). It is possible that one intron copy is excised from mtDNA by recombination to yield a circular intron DNA. PCR primers used to detect novel junctions resulting from ectopic insertions and the ensuing recombination events are indicated below the diagrams.

Figure 2

PCR analysis of retrotransposition into ectopic sites. (A and B) PCR assay for aI1 insertions into the E1/aI1 and 5β848 sites. mtDNAs (≈50 and 100 ng) from the indicated strains were analyzed by PCR to detect products of ectopic transposition into the E1/aI1 site (A; 22 cycles with primers P1 and P3) and 5β848 site (B; 25 cycles with primers P1 and P2). Similar results for the 5β848 site were obtained by using primers P3 and P4 (25 cycles) (not shown). Strain 1⁰2⁰ (lanes 9 and 10) has mtDNA identical to that of strain 1^+t2⁰ except that aI1 is deleted. (C) Estimate of the frequency of ectopic transposition events at the 5β848 site. mtDNA from strain 1^+t2⁰ (100 ng) was subject to 25 cycles of PCR using primers P1 and P2 to detect the downstream junction between aI1 and aI5β (see Fig. 1Bi), and that signal (lane 1) was compared with a calibration curve obtained with mtDNA from strain C2107, which contains the COXI deletion shown in Fig. 1Bii (lanes 2–9). In the experiment shown, decreasing amounts of mtDNA from strain C2107 were mixed with mtDNA from the inactive strain 1^YAHH2⁰ to maintain a constant 100 ng of DNA, and the same result was obtained in reaction mixtures containing only the C2107 mtDNA dilution series (not shown). A 5′-labeled 100-bp ladder was used for molecular weight markers (M) here and in the subsequent figures.

Figure 3

Diagram of wild-type and modified 5β848 sites and Northern hybridization analysis of sense- and antisense-strand transcripts of aI5β. (A) Diagrams of wild-type and constructed 5β848 alleles. The wild-type (W) strain contains the normal 5β848 site and lacks aI5γ (5γ⁰). The tagged wild-type 5β848 site (WT), containing flanking BamHI and NheI sites, was constructed by site-directed mutagenesis. Then the 50 bp between the BamHI and NheI sites were inverted (with disruption of the BamHI site) to make the inverted 5β848 site (INV). Both mutated sites were transformed into mtDNA to yield strains with the tagged WT or INV 5β848 sites. (B–D) Northern hybridizations. Northern blots of two independent preparations of cellular RNAs from the indicated strains were hybridized with a COXI exon 6-specific probe to detect COXI mRNA (loading control) (B) or oligonucleotide probes complementary to the sense (C) and antisense (D) strands of the 5β848 ectopic site. Strain 5β⁰ contains a COXI gene lacking aI5β. Faint signals at ≈2.3 and 3.4 kb in all strains are due to cross-hybridization with rRNA.

Figure 4

The inverted 5β848 site is a target for aI1 retrotransposition. The diagrams at the top show the expected products of aI1 retrotransposition into the tagged wild-type (WT) and inverted (INV) orientations of the 5β848 site. Insertion of aI1 in the sense orientation is detected by PCR using primers P3 and P4 (a) and P1 and P2 (b), whereas insertion of aI1 in the antisense orientation is detected by using primers P1 and P4 (c) and P2 and P3 (d). The gels show the products obtained in these reactions when mtDNA from strains with the WT and INV 5β848 sites was used. Reaction a used 27 cycles; reactions b and c used 25 cycles; and reaction d used 32 cycles.

Figure 5

Retrotransposition mechanisms using DNA targets. The COXI gene of strain 1^+t2⁰ (top) contains both the donor aI1 intron (hatched) and the 5β848 ectopic site in intron 5β (open rectangle) The mechanism on the left begins with reverse splicing into the ectopic site in double-stranded DNA. Inefficient nicking of the antisense strand forms the primer for full-length cDNA synthesis by the RT with completion of intron insertion by DNA repair. The mechanism on the right begins with reverse splicing into the ectopic site at a replication fork. cDNA synthesis is initiated either de novo or by using the 3′ end of the newly made leading strand with further replication and repair needed to complete intron insertion. Potential single-stranded DNA target sites may also exist on the lagging strand of the replication fork or in actively transcribed regions of mtDNA.