Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition.

LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.

LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein.

LINE-1 is a highly successful, non-LTR retrotransposon that has played a leading role in shaping mammalian genomes. These elements move autonomously through an RNA intermediate using target-primed reverse transcription (TPRT). L1 encodes two essential polypeptides for retrotransposition, the products of its two open reading frames, ORF1 and ORF2. The exact function of the ORF1 protein (ORF1p) in L1 retrotransposition is unknown, although it is an RNA-binding protein that can act as a nucleic acid chaperone. Here, we investigate the requirements for these two activities in L1 retrotransposition by examining the consequences of mutating two adjacent and highly conserved arginine residues in the ORF1p from mouse L1. Substitution of both arginine residues with alanine strongly reduces the affinity of the protein for single-stranded nucleic acid, whereas substitution of one or both with lysine has only minimal effects on this feature. Rather, the lysine substitutions alter the delicate balance between the ORF1 protein's melting and reannealing activities, thereby reducing its nucleic acid chaperone activity. These findings establish the importance of the nucleic acid chaperone activity of ORF1p to successful L1 retrotransposition, and provide insight into the essential properties of nucleic acid chaperones.