Mechanisms of non-LTR retrotransposition

The LINE-1 (L1) elements are a family of non-LTR retrotransposons that infest mammalian genomes. L1 sequences are present at nearly 1,000,000 copies in the human genome, and are directly or indirectly responsible for over 30% of human sequence (and probably more, given that ancient copies are unrecognizable due to mutation). 80% of human genes contain at least one L1 insertion, and many instances have been described where an L1 insertion has caused human disease. Despite these staggering statistics, we are largely ignorant of the processes that govern non-LTR retrotransposon replication, at both the regulatory and mechanistic levels. It is also unclear what other role, if any, that L1 plays in the evolution of our genetic material other than adding unnecessary bulk to our DNA sequence. Because of the continual bombardment of our genomes (and the genomes of many other species) with new mutations due to L1-like elements, we have an interest in understanding how they replicate and how new insertions may functionally affect the host genome. In addition, we plan to explore interactions between the host organism and the process of non-LTR retrotransposition.

A depiction of a full-length L1 element is shown in Figure 1A. The 5’ untranslated region (UTR) contains an internal promoter which begins transcription at base #1. This is followed by two open reading frames, ORF1 and ORF2. ORF1 encodes a nucleic-acid binding protein whose functional significance is unknown. ORF2 encodes a protein with endonuclease and reverse transcriptase activities, which are both essential for retrotransposition. A short 3’ UTR is followed by a polyA signal and tail, and the entire element is flanked by variable length target site duplications (TSDs). A rough outline of the L1 life cycle is shown in figure 1B, although the molecular details of each step (with the exception of TPRT, the initiation of first strand synthesis) remain a mystery. This process of retrotransposition continues to take place in all of us to this day.

In order to genetically dissect out the factors involved in the non-LTR retrotransposon life cycle, we are developing a model for retrotransposition in budding (S. cerevisiae) and fission (S. pombe) yeast. The molecular tools available and the ease of genetic manipulation of these organisms make them ideal for such a project. As L1 elements are absent in budding yeast, we have transplanted re-engineered versions of L1-like elements to recapitulate the transposition reaction. This will provide an assay to allow us to perform a variety of screens to identify genes responsible for the core mechanisms of L1 replication.