Mammalian L1 retrotransposon - host interaction

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RECENT FINDINGS: A largely unknown but major issue in L1 and mammalian biology is the mutual interaction between L1 & its host. We are examining two possible examples: (1) Adaptive evolution of ORF1p ORF1p is one of the two L1 encoded proteins essential for retrotransposition. ORF1p underwent adaptive evolution in recent primate history, an event that often implies a biological interaction (e.g., a virus & its host). To identify possible host targets of this event we used the yeast 2-hybrid assay to screen for host proteins that interact with the adapted ORF1p of the currently active human L1Pa1 family but not with its pre-adapted ancestral version, which we resuscitated from the L1Pa5 family. Repeated testing in the yeast 2-hybrid assay confirmed the interaction of five host proteins with ORF1p but did not reproducibly show a robust difference between their interaction with the adapted and pre-adapted ORF1ps. Also, evolutionary analysis showed that the 5 host proteins were highly conserved & had not undergone an adaptive response. However, a mammalian two-hybrid assay showed that some of the host proteins strongly interfere with multimer formation by ORF1p monomers. As multimer formation is necessary for ORF1p activity, an interaction between ORF1p and the host proteins could potentially be detrimental to both the host and L1. We are now determining whether the encoded host proteins inhibit retrotransposition. (2) L1 RNA transport - Others showed that the nuclear exchange factor (NXF1) binds a region of L1 RNA. NXF1 is a host protein that shuttles RNAs in & out of the nucleus. Evolutionary analysis showed that the L1 NXF1-binding site (part of which does not encode protein) has been highly conserved over 60 Myr of L1-evolution in primates. Preliminary experiments in collaboration with Marie-Louise Hammarskjld now show that NXF1 does indeed bind L1 RNA and we are now determing the structural requirements for this interaction. Before we test the requirement for NXF1 in retrotransposition we need to modify the current vector for measuring L1 retrotransposition as it likely bypasses the NXF1-mediated RNA transport pathway.