Reverse Transcriptase and Reverse Splicing Activities Encoded by the Mobile Group II Intron COBI1 of Fission Yeast Mitochondrial DNA

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Abstract

Mobile group II introns encode multidomain proteins with maturase activity involved in splicing and reverse transcriptase (RT) and (often) endonuclease activities involved in intron mobility. These activities are present in a ribonucleoprotein complex that contains the excised intron RNA and the intron-encoded protein. Here, we report biochemical studies of the protein encoded by the group IIA1 intron in the cob gene of fission yeast Schizosaccharomyces pombe mitochondria (cobI1). RNP particle fractions from the wild-type fission yeast strain with cobI1 in its mtDNA have RT activity even without adding an exogenous primer. Characterization of the cDNA products of such reactions showed a strong preference for excised intron RNA as template. Two main regions for initiation of cDNA synthesis were mapped within the intron, one near the DIVa putative high-affinity binding site for the intron-encoded protein and the other near domain VI. Adding exogenous primers complementary to cob exon 2 sequences near the intron/exon boundary stimulated RT activity but mainly for pre-mRNA rather than mRNA templates. Further in vitro experiments demonstrated that cobI1 RNA in RNP particle fractions can reverse splice into double-stranded DNA substrates containing the intron homing site. Target DNA primed reverse transcription was not detected unless a DNA target was used that was already nicked in the antisense strand of exon 2. This study shows that S. pombe cobI1 encodes RNP particles that have most of the biochemical activities needed for it to be a retroelement. Interestingly, it appears to lack an endonuclease activity, suggesting that the active homing exhibited by this intron in crosses may differ somewhat from that of the better-characterized introns.

Introduction

Reverse transcriptases (RT) are involved in various genetic phenomena. Phylogenetic comparisons of RT sequences reveal strong similarities, with seven highly conserved sequences,1 which are present in long terminal repeat (LTR) retroposons, non-LTR retroposons, telomerases, prokaryotic retrons, mitochondrial plasmids and group II introns.2 Nearly all group II intron-encoded proteins are closely related and the majority of each protein is encoded within domain IV of the intron RNA.3., 4. These proteins usually contain amino acid sequences that are closely related to (i) RTs,5 (ii) a family of RNA maturases6 and (iii) HNH endonucleases.7

Studies of the homing mechanisms of two bakers yeast group II introns (aI1 and aI2) and the Lactococcus lactis group II intron Ll.LtrB showed that group II intron mobility can be achieved by distinguishable pathways that may coexist.8 The intron-encoded protein (IEP) first binds to the unspliced precursor RNA forming a ribonucleoprotein (RNP) particle. It promotes splicing of the intron RNA and remains bound to the excised intron RNA lariat. In a homing cross, the DNA target is apparently first engaged by the DNA-binding domain of the IEP and sense strand of the target site is cleaved by partial or complete reverse splicing of the intron RNA followed by cleavage of the antisense strand downstream from the intron insertion site by the endonuclease activity of the IEP.9 In a reaction known as target DNA-primed reverse transcription (TPRT), the 3′-end of the cleaved antisense strand serves as the priming site for first-strand cDNA synthesis.9., 10., 11., 12. Finally, completion of the intron integration process is accomplished by one or more of several repair or recombination mechanisms using partial or full-length cDNA.3., 8., 13.

Four different reverse transcriptase assays have been described using RNP fractions enriched from lysates of bakers yeast (S. cerevisiae) mitochondria by centrifugation through a sucrose cushion. Similar assays have been used with similarly enriched RNP particles from Escherichia coli cells expressing the Lactococcus intron, but most of the published experiments for that system used RNP particles that were reconstituted from recombinant protein and self-spliced intron RNA lariat.14 The “endogenous assay” measures cDNA synthesis using endogenous RNA templates present in the RNP fraction.10 Although the wild-type aI1-encoded and aI2-encoded proteins are readily recovered in RNP particle fractions that have other relevant activities, aI1 lacks this endogenous RT activity completely13 and aI2 has a very low level of activity;10., 15. however, aI2 RNP particle fractions from several mutant strains exhibit substantial endogenous RT activity.16., 17., 18.

The P714T mutation of aI2 alters an amino acid at the boundary between the non-conserved D domain and the conserved En domain of the aI2 open reading frame.15 That mutation does not inhibit splicing but reduces the level of homing to various degrees, depending on the recipient allele used.8., 15. Studies with RNP particles from P714T mutant strains and an optimal DNA substrate showed that the P714T mutation partially inhibits the reverse splicing and antisense strand cleavage activities.8 By an unknown mechanism, it strongly activates the endogenous RT activity; the main template used in that reaction is intron RNA and a minor template is pre-mRNA. Mapped initiation sites are clustered in two main areas; around DVI on intron RNA and within exon 3 on pre-mRNA.10 This observation, together with the finding that deletion of domain V from the intron RNA stimulates this activity, suggests that altered RNA–protein interactions lead to alternative priming modes for the RT.18

Three other RT assays are useful with one or both of the S. cerevisiae introns. In the exogenous substrate assay, the endogenous RNAs in RNP particle fractions are degraded with RNase A and replaced by an oligo(dT)-primed poly(rA) substrate; various aI2 alleles have substantial activity but there is no activity for any aI1 strain tested.17., 19. The TPRT assay involves coupling the reverse splicing and endonuclease activities to the RT activity so that the cleaved antisense strand of a DNA substrate primes cDNA synthesis; both aI1 and aI2 are active in this assay.11., 19. Finally, a primer-dependent RT assay is effective for both of those introns (our unpublished results).17 Here, added oligonucleotides complementary to intron and flanking exon sequences in endogenous RNAs prime cDNA synthesis, but the most active primers mimic the 3′-end of the cleaved antisense strand.

The mitochondrial introns aI1 and aI2 of bakers yeast and the Lactococcus LtrB intron are the most extensively studied mobile group II introns, but they represent a small minority of the group II introns identified in nature. Although studies of the budding yeast group II introns helped to illuminate details of the homing mechanism, those two introns are related rather closely and have very similar homing pathways; as noted above, however, their RT activities differ somewhat. Comparing research on homing mechanisms by aI1, aI2 and the Lactococcus intron, it is evident that initial steps of retrohoming are highly conserved but downstream steps can vary considerably from intron to intron, and even for a given intron when different target sites or intron alleles are compared.

Here, we introduce fission yeast Schizosaccharomyces pombe (S. pombe) as a new system for investigating homing mechanisms of mitochondrial group II introns. Mitochondria of S. pombe strains contain small genomes with sizes ranging from 17.4 kb to 25 kb.20 Most of that variation is due to different numbers of group I and group II introns. A screen for the distribution of mitochondrial introns in the species identified group II introns in alleles of the cox1, cox2 and cob genes. All three group II introns, cox1I1a, cox2I1 and cobI1, belong to the A1 subgroup but are otherwise related only distantly.4., 21., 22., 23. The 2526 bp group IIA1 intron in the cob gene was first identified in strain 50 from the Leupold collection24 and it does not self-splice in vitro.21 The intron is mobile in crosses25 and exhibits ectopic homing.26

Here, we report initial biochemical characterization of the protein encoded by the cob intron. Our findings show that this intron encodes RNP particles that have most of the biochemical activities needed for it to be a retroelement resembling other mobile group II introns. Interestingly, the RNP particles appear to lack an endonuclease activity, suggesting that the very active homing mechanism used by this intron may differ somewhat from that of the better-characterized introns.

Section snippets

RNP particle fractions from S. pombe mitochondria

cobI1 of S. pombe mtDNA contains a long open reading frame that encodes a protein with amino acid segments related to reverse transcriptase, maturase and endonuclease domains of other group II introns. As indicated in Figure 1, the protein appears to be translated from the cob pre-mRNA, starting from the AUG start codon of cob exon 1 and terminating at the UAG stop codon near the end of the intron. Although the proteins encoded by introns aI1 and aI2 of budding yeast appear to be translated as

Discussion

Previous genetic studies showed that S. pombe cobI1 is mobile. In crosses between donor strain R10 and recipient strain P3, cobI1 was inserted at the junction between E1 and E2.25 The overall frequency of homing by this intron is 70–84%, similar to the 80–90% homing obtained with both bakers yeast introns.8., 13. The domain X mutation in mutant strain R10/5 was found to block both splicing and homing, and here we show that it inhibits RT activity significantly. Although RT-independent homing has

Strains

S. pombe strain R10 was constructed by elimination of all group I introns from the mtDNA of strain anar-14 and the intronless strain P3 was constructed by elimination of the cob intron from the mtDNA of strain R10.21 Strain R10/5 is a cob intron mutant of strain R10. A point mutation at nucleotide position 2734 changes a proline residue to serine close to the end of domain X of the cob intron ORF.21 Finally, strain 6G6 is a ρ° derivative of strain P3.27

Preparation of mtRNP particles

Fission yeast strains were grown in

Acknowledgements

The skilled technical assistance of Mrs Annette Schreer is acknowledged. This research was supported by NIH grant GM31480 and by grant I-1211 from the Robert A. Welch Foundation, both to P.S.P., and by a grant from the Deutsche Forschungsgemeinschaft (DFG) to K.W. and B.S.

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