Abstract
LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ∼34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35–99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ∼50% if overexpressed A3C is present.
Highlights
LINE-1 (L1) retrotransposon activity can cause disease by insertional mutagenesis, recombination, providing enzymatic activities for other non-long terminal repeat retrotransposons, and perhaps by transcriptional over-activation and epigenetic effects [reviewed in [1,2]]
To elucidate the mechanism that is responsible for the restriction of L1 retrotransposition by A3C [reviewed in [23]], we first investigated whether an intact cytidine deaminase (CDA) domain was required for A3C-mediated L1 inhibition
Mutating the Zn2+-coordinating Cys residues at position 97 and 100 to Ser (C97S/C100S) did not affect A3C-mediated L1 restriction significantly (P = 0.328, Figure 2A and Supplementary Table S1), and mutating both Cys residues separately even slightly increased the L1 inhibition from 54 to 87% (P = 0.0023) or 71% (P = 0.0349), respectively (Figure 2C and Supplementary Table S1). These findings clearly provide evidence that deaminase activity is not required for A3C-mediated L1 restriction
Summary
LINE-1 (L1) retrotransposon activity can cause disease by insertional mutagenesis, recombination, providing enzymatic activities for other non-long terminal repeat (nonLTR) retrotransposons, and perhaps by transcriptional over-activation and epigenetic effects [reviewed in [1,2]]. Nucleic Acids Research, 2014, Vol 42, No 1 397 endonuclease may have a general function in facilitating chromosomal breaks and genome instability [5,6]. To limit such deleterious effects of retrotransposition, host genomes have adopted several strategies to curb the proliferation of transposable elements. Mechanistic strategies used by the host to restrict the mobilization of transposable elements include DNA methylation [7,8,9], smallRNA–based mechanisms [10,11,12], DNA repair factors [13,14] and L1 restriction by TREX1 DNA exonuclease [15] and members of the human APOBEC3 (Apolipoprotein B mRNA Editing Enzyme Catalytic Polypeptide 3, A3) protein family of cytidine deaminases [reviewed in [16]]
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