RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins that are essential for retrotransposition. We earlier showed that primate ORF1p (like that in mouse) is a coiled coil mediated trimer that has nucleic acid binding and chaperone activity. However, the function of ORF1p in retrotransposition is largely unknown. In 2016 we had shown that L1 retrotranposition requires phosphorylation of ORF1p, reviewed in Furano, A. V. and P. R. Cook (2016). The challenge of ORF1p phosphorylation: Effects on L1 activity and its host. Mob Genet Elements 6(1): e1119927. For her last year here, Dr. Cook and I concurred that she pursue this issue independently. She found that the mitogen-activated protein kinase, p38delta, can phosphorylate ORF1p in vitro, which she published along with other findings. Also in 2016, our collaboration with the Williams laboratory (Northeastern Univ.) had shown that rapid oligimerization of ORF1p trimers on single stranded nucleic acid is essential for retrotransposition. Rapid oligomerization of ORF1p trimers (but not trimer formation per se) can be very sensitive to the amino acid sequence of the coiled coil, and this coiled coil property has been highly conserved for at least the last 30 Myr of primate evolution despite its repeated and extensive remodeling. In order to understand the causes and mechanisms of such dramatic change we carried out extensive phylogenetic and bioinformatic analysis of the ORF1 sequences of the seven L1 families, which emerged and then went extinct during this time period: L1Pa7-L1Pa1, the latter of which is currently active in humans. The results unexpectedly showed that multiple versions of the coiled coil region of ORF1p coexisted during the lifetime of each of the L1Pa7-L1Pa3 families. ORF1p heterogeneity only involved the coiled coil domain, which basically evolved as quasi-species which differed at multiple residues. One explanation for these unexpected results is that multiple amino acid substitutions could be required to compensate (suppress) certain deleterious mutations in a given heptad. The known extensive intra- and inter-strand cross-talk between the coiled coil heptads would be consistent with such a mechanism, which we have corroborated with experimental evidence. Such concerted amino acid changes are consistent with positive selection (more amino acid substitutions than expected by chance), which, in this case, is driven by the structural exigencies of the coiled coils rather than an adaptive response, as has been demonstrated for other instances of positive selection. These findings not only provide important insights on the processes that ensured the survival of the deleterious L1 parasite in primate genomes, but also on mechanisms of coiled coil evolution a widespread and important structural motif of many proteins.

Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2017
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
Type
DUNS #
City
State
Country
Zip Code
Furano, Anthony V; Cook, Pamela R (2016) The challenge of ORF1p phosphorylation: Effects on L1 activity and its host. Mob Genet Elements 6:e1119927
Naufer, M Nabuan; Callahan, Kathryn E; Cook, Pamela R et al. (2016) L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling. Nucleic Acids Res 44:281-93
Naufer, M Nabuan; Furano, Anthony V; Williams, Mark C (2015) 12 Oligomerization kinetics of ORF1p is correlated with LINE1 retrotransposition. J Biomol Struct Dyn 33 Suppl 1:7-8
Cook, Pamela R; Jones, Charles E; Furano, Anthony V (2015) Phosphorylation of ORF1p is required for L1 retrotransposition. Proc Natl Acad Sci U S A 112:4298-303
Callahan, Kathryn E; Hickman, Alison B; Jones, Charles E et al. (2012) Polymerization and nucleic acid-binding properties of human L1 ORF1 protein. Nucleic Acids Res 40:813-27