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.

FIG. 1.

Application of ligation-mediated PCR to amplify the 3′ ends of plus-strand cDNA. The oligonucleotide Rag208 blocked at its 3′ end with a C3 spacer (*) was ligated specifically to the 3′ ends of the cDNA. The oligonucleotide HL973 was complementary to Rag208 and was used with HL875 to PCR amplify the ligated products. The resulting fragment was inserted into the pCR2.1 TOPO vector (Invitrogen) and sequenced. The primer sites for synthesis of the minus strand (PBS) and the plus strand (PPT) are shown at the junctions of the LTRs and the body of the element.

FIG. 2.

The 3′ ends of the plus-strand cDNA. Ligation-mediated PCR was used to sequence the 3′ ends of plus-strand cDNA produced by wild-type Tf1-neoAI and the Tf1-neoAI elements with the mutations N782S and L783I in RNase H. The numbers of clones that terminated at each position within a 70-bp window are shown in histograms. The clones were categorized by the last nucleotide templated by the Tf1 sequence. N indicates the total number of clones graphed. (A) Wild type. (B) N782S. (C) L783I.

FIG. 3.

Recombinant Tf1 IN proteins and their ability to process the self primer from substrate oligonucleotides. (A) The substrates mimicked the U5 end of the LTR, and the sequences in boldface are the complement of the self primer (top strand) or the self primer itself (bottom strand). In addition to the wild-type Tf1 IN, we also tested a version of Tf1 IN lacking its C-terminal domain (ΔCH). The preprocessed U5 substrate was S1. The substrate with a single-stranded complement of the self primer was S2, and the substrate with a double-stranded addition was S3. S4 was the substrate with the self primer present as an RNA annealed to its DNA complement. The ribonucleotides are in italics. (B) The products of the reaction mixtures incubated with IN and ΔCH were analyzed on 13.5% sequencing gels. The 5′ ends of the top-strand oligonucleotides were labeled with ³²P, and the product with a precise cleavage removing the self primer is indicated by the asterisk.