Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed

Abstract

Background: The pFOXC retroplasmids are small, autonomously replicating DNA molecules found in mitochondria of certain strains of the filamentous fungus Fusarium oxysporum and are among the first linear genetic elements shown to replicate via reverse transcription. The plasmids have a unique clothespin structure that includes a 5'-linked protein and telomere-like terminal repeats, with pFOXC2 and pFOXC3 having iterative copies of a 5 bp sequence. The plasmids contain a single large open reading frame (ORF) encoding an active reverse transcriptase (RT). The pFOXC-RT is associated with the plasmid transcript in a ribonucleoprotein (RNP) complex and can synthesize full-length (-) strand cDNA products. In reactions containing partially purified RT preparations with exogenous RNAs, the pFOXC3-RT has been shown to initiate cDNA synthesis by use of snapped-back RNAs, as well as loosely associated DNA primers.

Results: The complete sequence of the distantly related pFOXC1 plasmid was determined and found to terminate in 3-5 copies of a 3 bp sequence. Unexpectedly, the majority of (-) strand cDNA molecules produced from endogenous pFOXC1 transcripts were attached to protein. In vitro experiments using partially purified pFOXC3-RT preparations having a single radiolabeled deoxyribonucleotide triphosphate (dNTP) generated a nucleotide-labeled protein that migrated at the size of the pFOXC-RT. The nucleotide preference of deoxynucleotidylation differed between pFOXC3 and pFOXC1 and showed complementarity to the respective 3' terminal repeats. In reactions that include exogenous RNA templates corresponding to the 3' end of pFOXC1, a protein-linked cDNA product was generated following deoxynucleotidylation, suggesting that reverse transcription initiates with a protein primer.

Conclusions: The finding that reverse transcription is protein primed suggests the pFOXC retroplasmids may have an evolutionary relationship with hepadnaviruses, the only other retroelement family known to initiate reverse transcription via a protein primer. Moreover, the similarity to protein-primed linear DNA elements supports models in which the terminal repeats are generated and maintained by a DNA slideback mechanism. The ability of the pFOXC-RT to utilize RNA, DNA and protein primers is unique among polymerases and suggests that the pFOXC plasmids may be evolutionary precursors of a broad range of retroelements, including hepadnaviruses, non-long terminal repeat (non-LTR) retrotransposons and telomerase.

Figure 1

Schematic diagram of the pFOXC plasmids. The pFOXC retroplasmids are approximately 1.9 kb linear, double-stranded DNA molecules that have a clothespin structure. They possess a covalently closed hairpin and iterative terminal repeats (black boxes) with a 5'-linked protein (circle). The plasmids have a single open reading frame (ORF) encoding a reverse transcriptase (RT; open box). The location of conserved domains characteristic of reverse transcriptases is indicated by shaded regions 1, 2, 2a, 3-7, with domain 2a also being conserved among non-long terminal repeat (non-LTR) retrotransposons. Details concerning the plasmid ORFs and iterative repeats are given in Table 1.

Figure 2

A portion of pFOXC1 and pFOXC3 endogenous reverse transcription products is associated with protein. (a) Mitochondrial ribonucleoproteins (mtRNPs) from pFOXC1-containing strains were untreated (lanes 1 and 4), or pretreated with actinomycin D (A; lanes 2 and 5) or RNAse A (R; lanes 3 and 6) for 5 min prior to reverse transcription reactions. Following precipitation, products were heated and electrophoresed in 1.2% agarose gels without (lanes 1-3) or with (lanes 4-6) 0.2% SDS in the gel and loading buffer. (b) Products from endogenous reverse transcription reactions pretreated with actinomycin D and using pFOXC1-containing (lanes 1-6) and pFOXC3-containing (lanes 7-9) mtRNPs were subjected to the following treatments: lane 1, no treatment; lane 2, incubation with proteinase K (K); lane 3, extraction with phenol-CIA (φ); lane 4, incubation with proteinase K followed by extraction with phenol-CIA (K, φ). Lanes 5 and 6 contain acetone-precipitated products recovered from the organic phase of the phenol extractions shown in lanes 3 and 4, respectively. Minus-strand cDNA products from pFOXC3-containing mtRNPs were subjected to the following treatments: lane 7, no treatment; lane 8, incubation with proteinase K followed by extraction with phenol-CIA (K, φ); lane 9, extraction with phenol-CIA (φ). Products were heated at 65°C in 0.2% SDS followed by electrophoresis in a 1.2% agarose gel containing 0.2% SDS. Marker sizes from 5'-end labeled λ-PstI restriction fragments are indicated in kb pairs on the left. The location of the wells is indicated with a gray arrow. The full-length (-) strand cDNA product is indicated on the right with a black arrow and a high molecular weight band detected in reactions lacking actinomycin D is indicated with an asterisk.

Figure 3

Protein-primed reverse transcription and identification of a nucleotide-linked protein in exogenous reverse transcription reactions. Mitochondrial ribonucleoprotein (mtRNP) particles were treated with micrococcal nuclease (MN), incubated with actinomycin D, and used in reactions having [α-³²P]dATP with various combinations of deoxyribonucleotide triphosphates (dNTPs), either in the absence or presence of an exogenous RNA, as indicated. (a) MN-treated pFOXC1-containing mtRNP particles were incubated in the absence (lane 1) or presence of a 92 nucleotide RNA corresponding to the 3' terminus of the pFOXC1 transcript with a full complement of nucleotides (lane 2). Reactions in lanes 3-5 were performed as in lane 2, but a single unlabeled nucleotide was omitted, as indicated. Products from exogenous reactions having a full complement of nucleotides and exogenous RNA were post-treated with proteinase K (K; lane 6), or extracted with phenol-CIA (φ; lane 7), prior to precipitation with ethanol. Products from a duplicate reaction of that in lane 2 were recovered from the aqueous (lane 8) and organic (lane 9) phase. (b) MN-treated pFOXC3-containing mtRNPs incubated in the absence (lanes 2-4) or presence (lanes 1, 5 and 6) of a 98 nucleotide RNA corresponding to the 3' terminus of the pFOXC3 transcript with [α-³²P]dATP and combinations of unlabeled nucleotides, as indicated. Products from a duplicate reaction of that shown in lane 5 were post-treated with proteinase K (K; lane 6). All reactions were boiled in Laemmli buffer prior to separation via 4-20% gradient SDS-PAGE. Prestained protein size markers are indicated on the left in kDa. Protein-linked products and (-) strand cDNA products are indicated on the right.

Figure 4

A nucleotide-linked protein comigrates with pFOXC3-reverse transcriptase (RT) and is unaffected by strong base treatment. Products of reverse transcription reactions using mitochondrial ribonucleoprotein (mtRNP) particles isolated from a pFOXC3-containing strain or a plasmid-free strain (P-F) having [α-³²P]dATP and unlabeled deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP) were separated via 10% SDS-PAGE and transferred to nitrocellulose. (a) The two panels on the left are from a nitrocellulose membrane probed with protein A-purified pFOXC3-RT_55-68 rabbit antiserum and visualized by chemiluminesence. The panel on the right is a phosphorimage of radiolabeled protein on the same nitrocellulose membrane. Prestained protein size markers are indicated on the left in kDa. The gray arrow indicates non-specific bands detected in the plasmid-free mtRNP preparation. (b) Micrococcal nuclease (MN)-treated pFOXC3-containing mtRNPs were incubated with [α-³²P]dATP and unlabeled dGTP and TTP. The products were separated via 4-20% gradient SDS-PAGE and radiolabeled products were detected by a phosphorimager. The gel was rehydrated in 1 M KOH and, following incubation at 55°C, the gel was neutralized and dried prior to detection by a phosphorimager.

Figure 5

Analysis of the nucleotide-linked protein in the presence of different combinations of deoxynucleotides and dideoxynucleotides. Micrococcal nuclease (MN)-treated pFOXC3-containing mitochondrial ribonucleoproteins (mtRNPs) were incubated with [α-³²P]dATP or [α-³²P]deoxyguanosine triphosphate (dGTP), and with unlabeled deoxyribonucleotide triphosphates (dNTPs) or dideoxynucleotide triphosphates (ddNTPs), as indicated. Reactions were terminated with Laemmli buffer and reaction products were separated via 4% to 20% SDS-PAGE. Prestained protein size markers are indicated on the left in kDa. (a) Reactions having [α-³²P]dATP or [α-³²P]dGTP with one unlabeled deoxynucleotide. (b) Reactions having [α-³²P]dATP or [α-³²P]dGTP, with a full complement of deoxynucleotides or dideoxynucleotides. (c) Reactions having [α-³²P]dGTP and a single dideoxynucleotide with a complement of deoxynucleotides, as indicated. (d) Reactions having [α-³²P]dATP and a full complement of deoxynucleotides incubated with or without phosphonoformate (PFA) at the indicated concentrations. Prestained protein size markers are indicated on the left in kDa and bands showing a slight size difference are indicated by an arrow.

Figure 6

A nucleotide-linked protein generated with [α-³²P]thymidine triphosphate (TTP) in pFOXC1-mitochondrial ribonucleoproteins (mtRNPs) is chased into a protein-cDNA product. (a) Micrococcal nuclease (MN)-treated pFOXC1-containing mtRNPs incubated with [α-³²P]dATP or [α-³²P]TTP and a full complement of deoxynucleotides, with or without a 92 nucleotide RNA template, as indicated. (b) MN-treated pFOXC1-containing mtRNPs incubated with [α-³²P]TTP with or without a 92 nucleotide RNA corresponding to the 3' end of pFOXC1. All four unlabeled deoxyribonucleotide triphosphates (dNTPs) were added to reactions to a final concentration of 20 μM at the times indicated. All reactions were terminated with Laemmli buffer and reaction products were separated via 4-20% gradient SDS-PAGE. Prestained protein size markers are indicated on the left in kDa.

Figure 7

Model for protein-primed reverse transcription by the pFOXC-reverse transcriptase (RT). Transcription of the pFOXC plasmid DNA molecules produces full-length RNAs that appear to function as both mRNAs for the synthesis of the RT and as templates for (-) strand cDNA synthesis [6]. Transcripts of pFOXC3 terminate in approximately three pentameric repeats, whereas transcripts of pFOXC1 terminate in approximately four copies of a 3 bp sequence (the 3' terminus of in vitro RNA used in this study is shown). Following production of the plasmid-encoded RT, deoxynucleotidylation occurs with the covalent addition of dAMP to a tyrosine residue of the 60 kDa pFOXC3-RT, followed by incorporation of deoxyguanosine monophosphate (dGMP) and a third nucleotide. Deoxynucleotidylation of the pFOXC1-RT results in the addition of thymidine monophosphate (TMP) to the RT, followed by one or more deoxynucleotide monophosphates (dNMPs) (a second TMP is shown). The resulting RT-(dNMP)_n complex would have complementarity to the corresponding terminal repeat. Based on studies of protein-primed DNA elements, the model predicts that the complex anneals to the penultimate 3' repeat of the template (shown for pFOXC1 only). Following the synthesis of a unit-length repeat, the RT-(dNMP)_n complex undergoes a slideback and is repositioned opposite the terminal repeat. The nascent cDNA is elongated via reverse transcription of the template by the 5'-linked RT or by a separate RT recruited to the complex. The model could also accommodate an increase in the number of repeats, depending on the number of slideback events that occur.