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Structure of a zinc-finger antiviral protein in complex with RNA reveals mechanism for selective targeting of CG-rich viral sequences

ABSTRACT: Infection of animal cells by many viruses is detected and countered by a variety of means, including recognition of non-self nucleic acids. The zinc-finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared to endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, four-zinc finger human (h) ZAP RNA binding domain (RBD), and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the four zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, crosslinking-immunoprecipitation-sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high affinity specific binding to CG-dinucleotide-containing RNA. Moreover, variations in the RNA binding specificity a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD:RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE139667 | GEO | 2019/11/01

REPOSITORIES: GEO

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Project description:RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the synthesis of non-coding RNAs by nuclear RNA polymerase E (NRPE or Pol V) 1-3, but the mechanism by which Pol V is targeted is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires the SU(VAR)3-9 homologs, SUVH2 and SUVH9. These proteins resemble histone methyltransferases, however a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-Adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of the DNA METHYLTRANSFERASE 1, MET1, causes losses of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. By tethering SUVH2 with a zinc finger to an unmethylated epiallele of the homeodomain transcription factor FWA, we demonstrate that SUVH2 is sufficient to both recruit Pol V and establish DNA methylation and gene silencing. Our results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of the plant genome for epigenetic silencing. For wild type plants (ecotype Columbia) and suvh2 suvh9 double mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. For the small RNA sequencing nrpd1 and nrpe1 mutant plants were sequenced in parallel as controls. For the bisulfite sequencing data, the wildtype data and that of the suvh2 and suvh9 single mutants was previously published and is thus not submitted here. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of wildtype and suvh2 suvh9 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPE1. Whole genome ChIP seq was also performed using a gifted endogenous NRPE1 antibody in a wildtype and met1 mutant background. Whole-genome bisulfite sequencing was also performed on fwa-4 epiallele plants transformed with the wild-type SUVH2 protein-coding construct containing a tethering zinc finger targeted to repeats at the fwa gene. For these transgenic libraries a line carrying a FLAG and fwa targeting zinc-finger tagged KRYPTONITE methyltransferase was bisulfite sequenced as a control (“FLAG-ZF-KYP”).

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Structure of a zinc-finger antiviral protein in complex with RNA reveals mechanism for selective targeting of CG-rich viral sequences

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