Chimeric RNA transposition intermediates of the I factor produce precise retrotransposed copies

Abstract

I elements in Drosophila melanogaster are non-long terminal repeat (LTR) retrotransposons of particular Interest because high levels of transposition can be induced by appropriate crosses. They use a full-length RNA transposition intermediate as a template for reverse transcription. Detailed molecular characterization of this intermediate is rendered difficult because of the many transcripts produced by defective elements. The use of an active I element marked with a sequence encoding the HA epitope solves this problem. We used an RNA circularization procedure followed by RT-PCR to analyze the transcripts produced by actively transposing tagged I elements. Most start at the 5′ end at the second nucleotide of the I element and all are polyadenylated at a site located in genomic sequences down-stream of the 3′ end. One of the tagged I elements, Inserted in locus 88A, produces chimeric transcripts that carry sequences from both 5′ - and 3′-flanking genomic DNA. We show that synthesis of these chimeric transcripts is controlled by the I element Itself. Analysis of full-length transposed copies of this element shows that the extra sequences at the 5′ and 3′ ends are not integrated during retrotrans-position. This suggest that initiation and arrest of reverse transcription during retrotransposition are precise processes.