Stem-loop structure preference for site-specific RNA editing by APOBEC3A and APOBEC3G.

Shraddha Sharma,Bora E. Baysal

doi:10.7717/peerj.4136

Abstract

APOBEC3A and APOBEC3G cytidine deaminases inhibit viruses and endogenous retrotransposons. We recently demonstrated the novel cellular C-to-U RNA editing function of APOBEC3A and APOBEC3G. Both enzymes deaminate single-stranded DNAs at multiple TC or CC nucleotide sequences, but edit only a select set of RNAs, often at a single TC or CC nucleotide sequence. To examine the specific site preference for APOBEC3A and -3G-mediated RNA editing, we performed mutagenesis studies of the endogenous cellular RNA substrates of both proteins. We demonstrate that both enzymes prefer RNA substrates that have a predicted stem-loop with the reactive C at the 3′-end of the loop. The size of the loop, the nucleotides immediately 5′ to the target cytosine and stability of the stem have a major impact on the level of RNA editing. Our findings show that both sequence and secondary structure are preferred for RNA editing by APOBEC3A and -3G, and suggest an explanation for substrate and site-specificity of RNA editing by APOBEC3A and -3G enzymes.

Highlights

The APOBEC3 (A3) family of cytidine deaminases restricts endogenous retroelements and exogenous viruses and plays an important role in the vertebrate innate immune system (Cullen, 2006; Chiu & Greene, 2008; Harris & Dudley, 2015)
Previous studies have shown that A3A-mediated DNA deamination of synthetic oligonucleotides occurs non- at TC dinucleotides (Chen et al, 2006; Shinohara et al, 2012; Sharma et al, 2015; Chan et al, 2015)
Bioinformatics analyses predicted that ∼98% of the edited Cs in A3G RNA substrates are located within secondary structures; the most common structure comprising of CNCC (N is any nucleotide) flanked by an average of four palindromic nucleotides (Sharma et al, 2016)

Summary

Introduction

The APOBEC3 (A3) family of cytidine deaminases restricts endogenous retroelements and exogenous viruses and plays an important role in the vertebrate innate immune system (Cullen, 2006; Chiu & Greene, 2008; Harris & Dudley, 2015). Recent studies suggest that A3 enzymes help retroviruses escape from drugs and adaptive immune recognition (Monajemi et al, 2012; Grant & Larijani, 2017). The A3 family comprises seven homologous enzymes in primates (Jarmuz et al, 2002; Conticello, 2008; Prohaska et al, 2014) that have either one (A3A, A3C and A3H) or two (A3B, A3D, A3F and A3G) zinc (Zn)-coordinating catalytic domains with HX1EX23–24CX2–4C motifs (X is any amino acid). The histidine and cysteine residues coordinate Zn2 + (Betts et al, 1994), and the glutamic acid residue may function as a proton shuttle during the deaminase reaction (Betts et al, 1994). Prior structural and biochemical studies have focused on the interaction of A3 enzymes and ssDNA oligonucleotides since C-to-U (C>U) deamination has been demonstrated in ssDNA exclusively. The A3 family members prefer a thymine immediately 5 to the target C, except APOBEC3G (A3G), which prefers a cytosine at the 5 position in

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Conclusion