The Self Primer of the Long Terminal Repeat Retrotransposon Tf1 Is Not Removed during Reverse Transcription

Abstract

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 5Ј ends of the cDNA so that their sequences are not copied into the 3Ј 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 3Ј 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)(12)(13)15). Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5Ј 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 5Ј 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 3Ј 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. 1). A ligation bias that could alter the sequence data obtained with this technique has not been observed (18). The ligation-mediated PCR ...

Single-Nucleotide-Specific Targeting of the Tf1 Retrotransposon Promoted by the DNA-Binding Protein Sap1 of Schizosaccharomyces pombe

“…This was tested using the homologous recombination assay, as described previously (Atwood-Moore et al 2006), which measures the amount of Tf1 cDNA in the nucleus by detecting homologous recombination between Tf1 cDNA and the Tf1 donor plasmid. In this assay, the neo gene within the Tf1 transposon is disrupted by an AI that renders it inactive until the intron is removed by splicing during transcription.…”

“…Preparation and analysis of nucleic acid cDNA preparations were performed as described previously (Atwood-Moore et al 2006). In brief, total DNA was extracted from cells grown for 36 hr using glass beads and phenol.…”

“…The polyclonal antisera used to detect Gag, integrase (IN), and reverse transcriptase (RT) have been described previously (Levin et al 1993;Hoff et al 1998). Preparation and analysis of nucleic acid cDNA preparations were performed as described previously (Atwood-Moore et al 2006). In brief, total DNA was extracted from cells grown for 36 hr using glass beads and phenol.…”

The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf 1 Is Stimulated by Tf 1 Integrase

“…Since in Tf1 the cellular tRNA is replaced by the SP, one could expect by analogy that the SP would also be removed by the RT-associated RNase H activity. Surprisingly, there is evidence that only a small fraction of the in vivo-generated Tf1 cDNA has the SP removed (2). This finding highlights another potentially exclusive property of the reverse transcription process of Tf1.…”

Template‐independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1

“…Sequence analyses of terminal sequences of the in vivo generated Tf1 cDNAs have also suggested that there are distinct features of this retroelement’s reverse transcription, such as the inefficient removal of the RNA self‐primer [11]. Based on these terminal sequences, substantial sequence additions, beyond the correct and conserved 3′‐end CA dinucleotides, were identified [11,12]. This may imply that Tf1 RT has an exceptional capacity to add non‐templated dNTPs to the cDNA ends.…”