Mammalian L1 retrotransposon - host interaction

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

RRECENT FINDINGS:[unreadable] ADATPIVE EVOLUTION OF ORF1p - ORF1p, which is an RNA chaperone, exists as a multimer, the monomer of which is tripartite. It consists of: an amino terminal domain of unknown function; a coiled-coil domain that is necessary for multimer formation; and a carboxyl terminal domain that contains a highly conserved nucleic acid binding domain. We previously showed that the coiled coil motif of ORF1p underwent episodes of adaptive evolution early in hominid evolution. Adaptive evolution often implies an interacting system (e.g., a virus & its host). Therefore, we used ORF1p as bait in a yeast two-hybrid screen for interacting mammalian proteins. After several rounds of screening we chose 5 proteins for further study: scaffold attachment factor A (safA), cold inducible RNA binding protein (CIRBP), heterogeneous nuclear RNA binding protein A1 (hnrnpA1), DEAD box polypeptide (URFH490), and lysyl tRNA synthetase. These proteins contain RNA-binding motifs or are known to bind RNA, the primary substrate for L1 retrotransposition. Experiments using both yeast and mammalian two-hybrid assays showed that these proteins interact differently with the ancestral (i.e., pre-adapted) and modern (i.e., adapted) versions of ORF1p. (See Z01 DK057601-11, for more information on the ancestral ORF1p). However, evolutionary analysis showed that all of these host proteins were highly conserved in primates. Thus, the adaptive evolution of ORF1p was apparently not a response to changes in these host proteins. We are now determining whether these host proteins differentially affect L1 retrotransposition promoted by L1 elements that encode either the modern (adapted) or ancestral (pre-adapted) ORF1p. As the tested host proteins serve essential functions for mammals, their putative interaction with L1 encoded proteins could contribute to the genetic load imposed on mammals by L1.[unreadable] L1 RNA TRANSPORT - Others showed that the nuclear exchange factor (NXF1, or Tap1) binds a region of the 3'UTR of L1 RNA. NXF1 is a host protein that mediates the export of non-spliced RNAs from the nucleus, as would be the case for L1 and retroviral RNAs. The dominance of L1 elements over other retrotransposons in mammals could result from L1 pre-empting NXF1. We found that the 3' UTR region that was reported to bind NXF1 has been highly conserved over 60 Myr of L1 evolution in primates. The strong conservation of a non-protein encoding sequence indicates that it must serve an essential function for L1 replication. However, in collaboration with Marie-Louise Hammarskjld we could not reproduce the original report of NXF1 interaction with this region of L1 RNA. We also tested the effect of deletions or other alterations of this region on retrotransposition in a cell culture based assay. These alterations had no effect. However, the current L1 retrotransposition assay vector contains several major modifications in the region of the 3'UTR, which may preclude its normal function. Therefore we are modifying the retrotransposition assay to eliminate these changes (see Z01 DK057601-11 LMCB: Mammalian L1 retrotransposon replication) and reexamine the role of the conserved region.