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.
Martin
Sandra L
SL
Department of Cell and Developmental Biology, University of Colorado School of Medicine, 12801 E. 17th Ave., Aurora, CO 80045, USA. sandy.martin@uchsc.edu
Cruceanu
Margareta
M
Branciforte
Dan
D
Wai-Lun Li
Patrick
P
Kwok
Stanley C
SC
Hodges
Robert S
RS
Williams
Mark C
MC
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008239:Lysine / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D048348:Reverse Transcription; D014157:Transcription Factors
C122171:Med14 protein, mouse; D018832:Molecular Chaperones; D011506:Proteins; D014157:Transcription Factors; D012313:RNA; D004247:DNA; D001120:Arginine; D008239:Lysine; D000409:Alanine