Multiple homing pathways used by yeast mitochondrial group II introns

Abstract

The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

FIG. 1

Sequences of recipient alleles and base-pairing interactions with the intron RNA. (A) Diagram of the aI2 target site and sequence differences among recipient strains. The diagram shows the region of the COXI gene containing the target site for aI2 homing. The exon sequence containing the aI2 target site of the S. cerevisiae donor strain is shown with sequence differences between it and the 1^o2^o Scap recipient allele containing COXI exons 1 to 3 derived from S. capensis indicated as larger boldface letters. Nucleotides are numbered according to their distance from the aI2 insertion site, which is between E2−1 and E3+1 (arrow and boldface vertical line). Other recipient alleles analyzed are derivatives of 1^o2^o Scap and are shown with nucleotide differences from the S. cerevisiae donor. Relevant sequence differences between donor and recipient COXI alleles outside of the region shown are indicated above the diagram. E1−387 is a small insertion containing a HpaII site that is present in the donor but not in the recipient allele. As indicated at the bottom, the recognition site for aI2 homing extends from E2−21 through E3+10 (5). (B) Effects of mutations on base pairing between the intron RNA and DNA target site. The diagram illustrates the known interactions between nucleotides of aI2 RNA and the sense strand of the aI2 target site. The conserved pairings IBS2-EBS2, IBS1-EBS1, and δ-δ′, spanning 7, 6, and 3 bp, respectively, are shown for the cross 1^o2⁺ × 1^o2^o Scap. Certain mutations alter one or another of those pairings, as shown. E2−8G improves the EBS2-IBS2 pairing, changing a C-A pair to C-G. Mutations E2−2T and E2−5C exchange G-T and G-C base pairs in IBS1-EBS1. E3+2T destabilizes the extended δ-δ′ interaction of the original donor-recipient pair.

FIG. 2

Analysis of aI2 homing in crosses. (A) Diagram of the cross between strains with the 1^o2⁺ and 1^o2^o COXI alleles. The donor (1^o2⁺) and recipient (1^o2^o) COXI alleles and the major 1^o2⁺ recombinant allele resulting from aI2 homing are diagrammed. Relevant restriction sites and the sizes of restriction fragments that are diagnostic for each parent and the major recombinant COXI allele are indicated. Not all progeny with the 1.8-kb allele have the same 3′ COXI features shown in the diagram; some also have introns aI5α, β, and γ due to aI5α homing and associated coconversion of aI5β and 5γ (16). The probe used hybridizes with the indicated part of exon 1. H, HpaII site; B, BamHI site. (B) Outputs of COXI alleles in crosses. Crosses between the indicated donor and recipient strains noted were carried out as described in Materials and Methods, and the output of COXI alleles was measured using the HpaII-BamHI digest diagrammed in panel A. Lanes 1 and 2 show the parental donor and recipient alleles, respectively. Lanes 3 to 10 show results of crosses between the pairs of strains indicated above each lane. Each lane contains DNA from mixed progeny of tens of thousands of matings so that the observed ratio of alleles reflects the extent of homing. Recipient strain ΔE3 is deleted for exon 3 (E3+1 through E3+35) and is a negative control in which the wild-type donor strain has no residual homing activity. Donor strain 1^o2^YAAA (lane 5) has missense mutations inactivating the aI2 RT activity. Sequence differences among the recipient strains in lanes 6 to 10 are defined in Fig. 1A. This gel is a representative outcome of these crosses, each of which was carried out at least in duplicate with analysis of two or more blots of each DNA sample. The same DNA samples were also scored for the output of alleles of the COB gene (as in reference 14), and the percentage of progeny with the recipient COB allele (COB-R) is indicated below the lanes. The percent COB-R alleles was used to calculate the percent homing, which is defined as the percentage of recipient COXI alleles that acquired aI2 by homing (see Materials and Methods for details about this calculation). The values shown below the lanes are for this gel, and mean values for each cross are provided in Table 3, column 1. A minor band present in some of the samples is due to cross-hybridization with other sequences in the DNA sample (compare lane 2 with lane 11).

FIG. 3

Assays of reverse splicing and antisense strand cleavage activities of RNP particles from strain 1^o2⁺. (A) Reverse splicing activity with different DNA substrates. Internally labeled DNA substrates were prepared from the recipient strains shown above each lane and incubated with RNP particles from strain 1^o2⁺, as described in Materials and Methods. Each lane contains the same number of counts of each substrate and the same amount of RNP particles. Size standards are shown to the left, and the substrate and the products of reverse splicing (RS) are identified to the right. The levels of reverse splicing products on this gel and four replicates were quantitated, and the mean values are reported in the text. (B) Antisense strand cleavage activity with different DNA substrates. DNA substrates from the indicated recipient alleles were made by PCR with a 5′-end label on the antisense strand, and antisense strand cleavage reactions were carried out with RNP particles from strain 1^o2⁺. The antisense strand is cleaved between nucleotides E3+10 and E3+11. The amount of cleavage product was quantitated on this gel and one repeat, and the values are reported in the text.

FIG. 4

Analysis of effects of the Zn domain mutation P714T on reverse splicing, antisense strand cleavage, and homing. (A) Reverse splicing. Internally labeled DNA substrates were prepared from the recipient strains shown above each lane and incubated with RNP particles from strain 1^o2⁺ (WT) and 1^o2^P714T (P714T), as described in Fig. 3A. Size standards are shown on the right, and the substrate and products of reverse splicing are identified on the left. The levels of products were quantitated, and the values are reported in the text. (B) Antisense strand cleavage. DNA substrates from the recipient alleles shown above the lanes were made by PCR with a 5′-end label on the antisense strand, and antisense strand cleavage reactions were carried out with RNP particles from the wild-type (WT) and P714T strains as in Fig. 3B. The amounts of cleavage product were quantitated, and the values are reported in the text. (C) Homing in 1^o2^P714T × 1^o2^o crosses. Crosses between the Zn domain mutant P714T donor strain and the indicated recipient strains were carried out and analyzed as in Fig. 2B. The RFLP alleles are as diagrammed in Fig. 2A. Lanes 1 and 2 contain DNA from the parental strains, and lane 3 contains DNA from the control cross between 1^o2⁺ × 1^o2^o E2−8G E3+2T+5G (cross E1, see Table 2). Data for cross E3 are shown in lanes 6 and 7. The parameters “% COB-R” and “% homing” are as defined in Fig. 2 and in Materials and Methods.

FIG. 5

Coconversion patterns associated with aI2 homing. The first two lines are diagrams of the aI2 donor and recipient alleles used in the crosses analyzed here. These crosses yield three major coconversion patterns in various proportions as discussed in the text. Pattern 1, in which there is efficient coconversion of markers in both 5′ and 3′ exons, results from the main pathway for RT-independent homing in which cleavage of the DNA target site is followed by DSBR (see also Fig. 6d). Pattern 2, in which there is efficient coconversion of markers in the 5′ exon but no coconversion in the 3′ exon, results from the major retrohoming pathway (Fig. 6b and c). Pattern 3, in which there is no coconversion associated with aI2 insertion, results from retrohoming events that are activated by the E2−8G and E3+2T mutations of the recipient strain (Fig. 6a). Also shown are three minor coconversion patterns that reveal a gradient of coconversion. Patterns 1a and 1b result from RT-independent homing events, while pattern 2a results from retrohoming events.

FIG. 6

Different pathways used for aI2 homing. The diagram summarizes features of the aI2 homing pathways analyzed in this study. The donor and recipient alleles are shown in the first line. Sense and antisense strands of the donor and recipient DNAs are identified, and the strand polarity is indicated by half arrowheads at the 3′ ends. White strands indicate recipient DNA exons, black strands indicate donor DNA exons, and thick gray strands indicate intron DNA. The donor strain synthesizes aI2 RNP particles containing excised intron RNA (thick dashed lines) and the intron-encoded RT protein. Homing is initiated by partial or full reverse splicing into the aI2 DNA target site. The next line shows the products of partial and full reverse splicing, which are intermediates in all of these homing pathways. Each intermediate can potentially be reverse transcribed by the intron-encoded RT to yield partial or full-length cDNAs (gray lines). In pathway a, a full-length cDNA synthesized from fully reverse spliced intron RNA leads to retrohoming via a repair process that does not appear to involve recombination. This pathway results in insertion of the intron into the recipient DNA with no coconversion of flanking exon sequences, as indicated at the bottom of the figure. In pathways b and c, incomplete cDNAs synthesized from fully or partially reverse spliced intron RNA complete retrohoming by using the mitochondrial DSBR recombination system are shown. The figure illustrates strand invasion of the donor DNA by the cDNA and completion of the intron DNA synthesis using the donor DNA as template with copying beginning in the intron and extending into the 5′ exon, followed by strand exchange back to the recipient DNA. Subsequent steps, including removal of the intron RNA and synthesis of the opposite DNA strand, are not shown. These events result in insertion of the intron with unidirectional coconversion of upstream exon sequences. In the RT-independent pathway d, a cleaved DNA target site containing partially reverse-spliced intron RNA leads to homing via gapping and strand invasion of the donor mtDNA. The same outcome could result from a cleaved DNA target site that initially contains a fully reverse spliced intron RNA (not shown, for simplicity). The RT-independent pathway results in insertion of the intron with coconversion of both upstream and downstream exon sequences, as in group I intron homing.