Abortive transposition by a group II intron in yeast mitochondria

Abstract

Group II intron homing in yeast mitochondria is initiated at active target sites by activities of intron-encoded ribonucleoprotein (RNP) particles, but is completed by competing recombination and repair mechanisms. Intron aI1 transposes in haploid cells at low frequency to target sites in mtDNA that resemble the exon 1-exon 2 (E1/E2) homing site. This study investigates a system in which aI1 can transpose in crosses (i.e., in trans). Surprisingly, replacing an inefficient transposition site with an active E1/E2 site supports <1% transposition of aI1. Instead, the ectopic site was mainly converted to the related sequence in donor mtDNA in a process we call "abortive transposition." Efficient abortive events depend on sequences in both E1 and E2, suggesting that most events result from cleavage of the target site by the intron RNP particles, gapping, and recombinational repair using homologous sequences in donor mtDNA. A donor strain that lacks RT activity carries out little abortive transposition, indicating that cDNA synthesis actually promotes abortive events. We also infer that some intermediates abort by ejecting the intron RNA from the DNA target by forward splicing. These experiments provide new insights to group II intron transposition and homing mechanisms in yeast mitochondria.

Figure 1.—

Diagrams of the standard aI1 donor and recipient COXI alleles, a putative intermediate and the main product of cis transposition. Exons and intron reading frames are tall and shorter rectangles, respectively. The 1^+t2⁰ donor and 1⁰2⁰ E1-6T recipient alleles (lines 1 and 2) differ in the number of introns and the presence or spacing of the key restriction enzyme sites (H, HpaII; B, BamHI; and R, EcoRI). Line 3 shows the structure of the putative twintron intermediate in cis transposition and line 4 shows the final product that results from recombination between the two intron copies in line 3. The respiratory ability (glycerol growth) of each strain is indicated. Before this study it was not known whether the intermediate would be Gly⁺ or Gly⁻. Exon 1 in the donor and recipient strains is colored differently to indicate that the exons differ at several nucleotides (Eskes et al. 1997, 2000); the nucleotide E1-6 is a T in these two strains but an inactive allele, E1-6A, is present in recipient strains in this study (see Figure 2).

Figure 2.—

COXI allele diagrams for the 1^+t2⁰ × aI5βE1/E2 cross. The donor COXI allele 1^+t2⁰ and recipient COXI alleles 1⁰2⁰ aI5β848 and 1⁰2⁰ aI5βE1/E2 are diagrammed in lines 1–3, respectively. The sequence of aI5β in the aI5β848 recipient strain (line 2) is the same as in the donor strain (line 1) except that an NheI site has been added 20 bp after the ectopic insertion site. The aI5βE1/E2 strain (line 3) contains 50 bp of E1/E2 sequence in aI5β that includes the aI1 homing site plus the NheI site that follows the 20 bp of E2. The sequence of the inserted E1/E2 is exactly the same as that of the natural E1 and E2 in the donor strain. Restriction sites that were assayed in DNA blots (Figure 3) are shown along with the lengths of the expected restriction fragments. Probes that hybridize to a sequence in the last exon (large arrow) or in exon 5β (open circle) were used as indicated. The recipient E1 sequence at the natural location has the E1-6A allele that blocks homing there (Eskes et al. 1997). Line 4 shows the expected twintron product of aI1 transposition into the ectopic target in the strain shown in line 3. The locations of PCR primers used to detect twintron products in mtDNA from this cross are indicated in the red box below line 4 (see Dickson et al. 2001). Line 5 shows the actual main product of transposition in this cross. Line 6 shows a retrodeletion that is a minor product of transposition. The respiratory phenotype of strains carrying each allele is indicated. X, XbaI; N, NheI; H, HpaII; B, BamHI; R, EcoRI.

Figure 3.—

RFLP analysis of transposition in crosses. Crosses between the indicated donor and recipient strains were carried out as described in materials and methods and the outputs of COXI alleles were measured using the XbaI+NheI digest diagrammed in Figure 2, hybridized with the exon 5β probe (open circle in Figure 2). The same gel was hybridized with the exon 6 probe (arrowhead in Figure 2) and the results are discussed in the text. Lanes 1 and 2 show the 1^+t2⁰ and 1⁰2⁰ 5βE1/E2 parental alleles and lane 4 shows the results of the cross between them. Lane 3 is DNA from a representative product of abortive transposition isolated from that cross (diagrammed in Figure 2, line 5). Lane 5 shows the result of a cross using an RT-deficient donor strain. Lanes 6–8 show results of crosses between the 1^+t2⁰ donor and recipient strains with the 5β848, E1/5β, and 5β/E2 targets, respectively. Lanes 9 and 10 show crosses using a recipient strain with the E1/E2 target inserted in aI5β in the inverse orientation and lane 11 shows the cross between donor strain 1⁺2⁰ and recipient 5βE1/E2. The DNA samples for all of the crosses were also scored for the output of alleles of the COB gene (not shown; see materials and methods and Eskes et al. 1997). The percentage of progeny with the recipient COB allele was measured and used to calculate the extent of abortive transposition in each cross (summarized in Table 1).

Figure 4.—

PCR analysis of twintron products of transposition. PCR reactions using primers defined in the red box below line 4 of Figure 2 and balanced amounts of mtDNA from the strains indicated above lanes 1–4 were carried out for 24 cycles and the products were fractionated on a 2% agarose gel and stained with ethidium bromide. The reaction in lane 1 contains 12.5 ng of purified mtDNA based on A260. Reactions in lanes 2–4 contain an equivalent amount of mtDNA based on quantitated Southern blots for common COB gene sequences. The identity of the PCR product in lane 1 was confirmed by DNA sequencing. To estimate the level of twintrons formed in the crosses shown in lanes 1 and 4, PCR reactions were carried out with the indicated dilutions of a balanced sample of mtDNA from a diploid strain that has the twintron allele (lanes 5–10).

Figure 5.—

Pathways for aI1 transposition. The diagram summarizes features of aI1 transposition demonstrated or inferred in this study (see also Figures 2 and 3). Line 1 shows the donor COXI allele 1^+t2⁰. Line 2 shows the recipient COXI allele aI5βE1/E2. Line 3 shows the initial retrotransposition intermediate formed by reverse splicing and antisense-strand cleavage at the ectopic E1/E2 target. It is a key intermediate that can be further processed in at least the three different ways indicated (from line 3 to line 4 or to line 6 or to line 11). Line 4 shows the intermediate in which an initial cDNA has been primed by the cleaved antisense strand of the target site. Line 5 shows a later intermediate in which the inserted intron RNA has been degraded; although the diagram shows the complete removal of the RNA, the most important aspect of this step is removal of the RNA complement of the cDNA. Line 6 shows the site after resection to remove the short cDNA and portions of aI5β flanking the cleaved target site; resection of both strands is likely but to progress to the next step it is likely that ends with 3′-ended single strands are formed. Some of the evidence suggests that some of the line 3 intermediate progresses directly to the one shown in line 6 by degradation of the inserted intron RNA. Line 7 illustrates the main product of abortive transposition formed from the intermediate shown in line 6 by strand invasion of aI5β sequences in a donor mtDNA (as in line 1). In some instances resection of the cleaved target extends into exon 6 (line 8) and that intermediate can invade the donor mtDNA, resulting in the repaired product shown in line 9. The intermediate in line 5 can invade aI1 sequences in a donor mtDNA with completion by recombination forming the deleted product of transposition shown in line 10. As developed in the text, the intermediate in line 3 may be repaired directly back to its original state by ejecting the inserted intron RNA by forward splicing, followed by ligation of the antisense strand (line 11).