RNA-Mediated Dimerization of the Human Deoxycytidine Deaminase APOBEC3H Influences Enzyme Activity and Interaction with Nucleic Acids

Abstract

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12 nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.

Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

“…While this disruption decreased catalytic activity, it did not cause protein unfolding. Rather, it decreased the interaction strength with a dsRNA molecule that A3H acquires from the cell and uses to dimerize 9,[47][48][49][50] . This dimerization using an RNA intermediate is essential to A3H thermodynamic stability 47,48 .…”

“…Rather, it decreased the interaction strength with a dsRNA molecule that A3H acquires from the cell and uses to dimerize 9,[47][48][49][50] . This dimerization using an RNA intermediate is essential to A3H thermodynamic stability 47,48 . While the nature of dimerization for different A3s is unique, e.g., only A3H uses an RNA intermediate, a SNP that regulates activity through dimerization has also been found for A3C 46,60 .…”

“…Although A3B activity can be lost through a gene deletion 44 , other A3s have lost activity because of decreased catalytic activity due to mutation of residues near the active site, e.g., A3D or loss of dimerization, e.g., A3C 45,46 . Based on recent crystal structures, the association of A3H with RNA is concomitant with enzyme stability and dimerization 9,[47][48][49][50] . This is a unique feature of A3H compared to other A3s, in that it uses a doublestranded RNA molecule to form a dimer interface without any protein-protein contacts and that this imparts stability to the enzyme.…”

“…Further, both the A3H Hap I K117E and K117E/K121E binding curves best fit to a rectangular hyperbola by least squares analysis, in contrast to A3H Hap VII and A3H Hap II that best fit to a sigmoid. The sigmoidal binding relationship for A3H Hap II has been shown to be due to further 15 oligomerization of A3H dimers on ssDNA 48 . This does not appear to occur with A3H Hap I K117E or K117E/K121E (Figure 5e-f), although they can form dimers in solution (Figure 5b-d).…”

Polymorphisms in human APOBEC3H differentially regulate ubiquitination and antiviral activity

“…Two A3H monomers interact with opposite sides of a short RNA duplex, and the A3H monomers in this complex interact primarily with the RNA duplex and not with one another. Importantly, mutagenesis of residues in the interaction interface between A3H and duplex RNA results in a decrease in A3H expression similar to the unstable human haplotypes [25][26][27]. Thus, formation of the A3H-RNA complex may be a critical regulator of A3H stability.…”