Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity

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.

Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein

“…L1 expression and retrotransposition are suppressed by many diverse cellular pathways in order to minimize the genomic damage inflicted by L1 activity [20,[43][44][45][46][47][48][49][50]. L1 encodes an ORF2 protein with several identified functions essential for the retrotransposition process.…”

miR-128 represses L1 retrotransposition by binding directly to L1 RNA

“…Somatic cells attenuate element mobilization by DNA methylation of the L1 promoter 23 . Other methods of regulation are mediated by APOBEC proteins 24,25 , microprocessor interactions 26 and Ago-mediated RNA interference in mouse embryonic stem cells 27 . L1-promoter silencing is greatly attenuated, and L1 transcription is reactivated in hypomethylated cells, such as cancer cells and tumor-initiating cells, and is also reactivated during reprogramming [28][29][30] .…”

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…First, since A3 enzymes bind RNA with high affinity, it was thought that the A3 enzymes would act as a roadblock to reverse transcription. Similarly, A3s could bind the LINE-1 RNA in the cytoplasm and cause it to accumulate in cytoplasmic RNA processing bodies (P-bodies), preventing nuclear import [167,168]. DNA sequencing of integrated LINE-1 genomes had little evidence of cytosine deamination, which supports these mechanisms mediated by RNA binding.…”

“…While A3G has weak deaminationindependent activity, other A3 enzymes, such as A3F and A3H, have potent deaminationindependent activity [74,154,169,286]. A3C restricts LINE-1 retroelements by deaminationindependent mechanisms [167,214]. Therefore A3C, and other A3s, may utilize deaminationindependent mechanisms in order to supplement the observed weak restriction activity (e.g., Figure 5.8).…”