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The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5′ end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer.
The reverse transcription of retroviruses and long terminal repeat (LTR) retrotransposons is primed by specific tRNA species for minus-strand initiation and by polypurine tracts (PPT) for plus-strand initiation. These primers play a critical role in defining the ends of the cDNA such that the “CA” dinucleotides required for integration are positioned at the termini (23). During reverse transcription the RNase H activity of reverse transcriptase (RT) removes the tRNA and PPT primers from the 5′ ends of the cDNA so that their sequences are not copied into the 3′ termini of the complementary strand of cDNA. This is a critical feature of reverse transcription because addition of these nucleotides after the conserved “CA” at the 3′ ends of the cDNA would block integration.
The LTR retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription (11-13, 15). Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5′ end of its own transcript. An increasing number of LTR elements in eukaryotes from yeast to vertebrates are found to use this self-priming mechanism (3, 16).
A recent study of mutations in the RT of Tf1 revealed that RNase H removes the PPT from the 5′ end of the plus-strand cDNA (2). Random mutagenesis of RT resulted in a cluster of mutations in RNase H that inhibited the removal of the PPT without reducing the amount of cDNA produced. That RNase H was responsible for primer removal was not surprising because the PPT of Tf1 is similar to those of other LTR elements. However, because of its unique nature, it is not known whether the self primer of Tf1 is also removed by RNase H.
To determine whether the self primer of Tf1 was removed during reverse transcription, we sequenced the ends of cDNA produced by the plasmid copy of Tf1 (Tf1-neoAI) used for transposition assays (1, 11). Virus-like particles were isolated from cultures of S. pombe (YHL6742) and purified on sucrose gradients, and cDNA was extracted (11, 14). We determined the sequence of the cDNA at the 3′ end of the plus strand by ligating an oligonucleotide to the cDNA and using a complementary oligonucleotide to amplify by PCR the terminal sequences (Fig. (Fig.1).1). A ligation bias that could alter the sequence data obtained with this technique has not been observed (18). The ligation-mediated PCR was conducted with conditions described previously (2). The PCR product amplified from the ligation reaction was inserted into the vector pCR2.1, and 124 clones were sequenced. As observed previously, the majority of the cDNA had 3′ termini that ended with one, two, or three untemplated nucleotides (2). Of the 79% of the clones that had untemplated nucleotides, 1% had one untemplated nucleotide, 68% had two untemplated nucleotides, and 15% had three untemplated nucleotides. The sequences of the untemplated nucleotides varied considerably, demonstrating that the clones resulted from many independent cDNA species.
The 3′ termini were mapped on the histogram in Fig. Fig.22 according to the position of the last templated nucleotide. By far, the most common cDNA contained the entire self primer (Fig. (Fig.2A).2A). The self-primer sequence found at the 3′ end of the plus stand was undoubtedly templated during reverse transcription by the primer itself, present at the 5′ end of the minus strand. These data demonstrated that the self primer had not been removed from this cDNA before RT completed the synthesis of the plus strand.
Although the cDNA with the full-length self primer was far more prevalent than any of the other species, it was formally possible that it represented a “dead-end” species that could not contribute to integration. If this is true, some of the minor cDNAs might have represented the active species from which the self primer was removed. We tested this possibility using the mutations in RNase H that were found previously to significantly inhibit the removal of the PPT (2). Such mutants might be expected to cause a similar defect in removing the minus-strand primer. The cDNA produced by Tf1 with the mutations N782S and L783I in RNase H was analyzed by ligation-mediated PCR, and the 3′ termini were mapped on the histograms in Fig. 2B and C. The cDNA profiles produced by the mutants were virtually identical to that of the wild type. No increases were observed in the levels of cDNA with the full-length self primer, and no increases were seen in cDNAs with incomplete portions of the self primer. Three other mutations in RNase H, I784T, R786C, and S749L, were previously found to cause defects in transposition but not in the removal of the PPT (2). The cDNAs produced by these mutants were also examined by ligation-mediated PCR, and, as expected, no increases in the species with the self primer were found (data not shown).
Our data indicate that the principal species of cDNA produced by wild-type Tf1 retained the self primer on the 5′ end of the minus strand and that RNase H did not remove this primer. These findings are particularly surprising because of how they differ from those on the processing of tRNA primers. Evidence from many laboratories demonstrates that the tRNA primers of both retroviruses and retrotransposons are efficiently removed by RNase H before reverse transcription of the termini is complete (4-6, 10, 17, 21, 23). The removal of the tRNA primers is essential for positioning the “CA” dinucleotide at the 3′ end of the plus strand, where it can be recognized by integrase (IN) (7). Although the processing activity of INs that removes nucleotides 3′ of the “CA” is capable of removing one or two nucleotides, it cannot remove the extensive sequences of the tRNA primers. Thus, cDNAs that retain the tRNA primers would be inactive for integration.
The presence of the self primer on the 3′ end of the cDNA suggests the possibility that Tf1 IN may have a novel processing activity capable of removing the 11-nucleotide primer. Consistent with this model is the recent finding that the IN of Tf1 possesses a processing function and that this activity is capable of removing several nucleotides (9). Experiments in Fig. Fig.33 tested whether recombinant IN was capable of removing the self primer from the 3′ ends of model substrates. The oligonucleotide substrates mimicked the U5 end of the LTR (Fig. (Fig.3A).3A). While we were able to detect precise removal of the intact self primer when present as a single-stranded DNA extension (Fig. (Fig.3B,3B, S2+IN), we were unable to detect specific removal of the self primer when present as a double-stranded extension of a DNA/DNA duplex (Fig. (Fig.3B,3B, S3+IN) or as an RNA/DNA duplex (Fig. (Fig.3B,3B, S4+IN). The possibility remains that RNase H and IN function together during integration to remove the complement of the self primer when annealed to the primer itself. A growing body of experimental evidence indicates that retroelement IN and RT proteins cooperate in various steps during their propagation (8, 10, 19, 20, 22, 24-26).
Although the large amounts of cDNA with the primer suggest the accumulation of an authentic intermediate, it is possible that this cDNA is not an active substrate for integration. The small levels of cDNA without the primer could account for the transposition observed.
The data here show that the self primer is retained at the 3′ end on the bulk of the plus-strand cDNA. Based on the dominant levels of cDNA with the primer, we propose the possibility that IN or IN with RT removes the primer.
Just prior to the publication of this article, we learned that the oligonucleotide HL1455, a component of substrate S4 in Fig. Fig.3,3, did not contain RNA nucleotides. Instead the 11 nucleotides at the 5′ end contained 2′ O-methyl nucleotides, an RNA analog. Since the experiment tested the ability of IN to cleave the DNA annealed to HL1455, the 2′ O-methyl modifications were not likely to have a direct impact on IN activity. However, the statement that we were unable to detect removal of the self-primer when present as an RNA/DNA hybrid should be interpreted with caution.
This research was supported by the Intramural Research Program of the NIH from the National Institute of Child Health and Human Development and the NIH Intramural AIDS Targeted Antiviral Program.