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. 2003 Mar 7;278(10):8112-7.
doi: 10.1074/jbc.M210487200. Epub 2002 Dec 27.

High-affinity, non-sequence-specific RNA binding by the open reading frame 1 (ORF1) protein from long interspersed nuclear element 1 (LINE-1)

Vladimir O Kolosha  1 , Sandra L Martin
Affiliations
Free article

High-affinity, non-sequence-specific RNA binding by the open reading frame 1 (ORF1) protein from long interspersed nuclear element 1 (LINE-1)

Vladimir O Kolosha et al. J Biol Chem. .
Free article

Abstract

Long interspersed nuclear element 1 (LINE-1 or L1) is an interspersed repeated DNA found in mammalian genomes. L1 achieved its high copy number by retrotransposition, a process that requires the two L1-encoded proteins, ORF1p and ORF2p. The role of ORF1p in the retrotransposition cycle is incompletely understood, but it is known to bind single-stranded nucleic acids and act as a nucleic acid chaperone. This study assesses the nature and specificity of the interaction of ORF1p with RNA. Results of coimmunoprecipitation experiments demonstrate that ORF1p preferentially binds a single T1 nuclease digestion product of 38 nucleotides (nt) within the full-length mouse L1 transcript. The 38-nt fragment is localized within L1 RNA and found to be sufficient for binding by ORF1p but not necessary, because its complement is also efficiently coimmunoprecipitated, as are all sequences 38 nt or longer. Results of nitrocellulose filter-binding assays demonstrate that the binding of ORF1p to RNA does not require divalent cations but is sensitive to the concentration of monovalent cation. Both sense and antisense transcripts bind with apparent K(D)s in the low nanomolar range. The results of both types of assay unambiguously support the conclusion that purified ORF1p from mouse L1 is a high-affinity, non-sequence-specific RNA binding protein.

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