REGULATION AND FUNCTION OF RETROELEMENTS

Child Health And Human Development

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Abstract

The mechanisms that retroviruses use to propagate can be effectively modeled by LTR-retrotransposons such as Tf1 of fission yeast. Despite the significant number of similarities that retrotransposons share with retroviruses, the RT of Tf1 uses a novel mechanism of self-priming to initiate cDNA synthesis. The first 11 bases of the Tf1 transcript anneal to the primer binding site and a cleavage reaction results in the production of the 11 nucleotide primer of reverse transcription. By co-expressing two distinct versions of Tf1, we found the unusual result that the first 11 nucleotides of one Tf1 mRNA were able to prime, in trans, the reverse transcription of other mRNAs. In other experiments we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others. These findings indicate that despite the novelty of its primer, the reverse transcription of Tf1 exhibits the same type of recombination as that of retroviruses. The presence of an intact nuclear envelope throughout the cell cycle of fission yeast is an important feature of this system that allows us to model the transport of HIV preintegration complexes (PICs) into the nuclei of nondividing cells. We found that nup124 encodes a nuclear pore factor that is required for transposition. To identify the function of Nup124p in the nuclear localization of Tf1, we have studied the nuclear localization activity of Gag. The localization of GFP-LacZ proteins fused to segments of Gag revealed that residues 10 to 30 of Gag, a segment that lacks an NLS, causes the import activity of the NLS to require Nup124p. The results of alanine scanning mutagenesis identified a segment of 4 amino acids in Gag that is necessary for Nup124p dependent import into the nucleus. What was particularly interesting was that mutations in these residues resulted in nuclear localization that no longer depended on Nup124p activity. Therefore, these specific residues in Gag appear to simply regulate transport. We are now testing these residues for modifications that could inhibit import by a unique mechanism that is mediated by Nup124p.