Abstract
Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform–PAZ–Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.
Highlights
Dicer belongs to the ribonuclease III (RNase III) family of double-stranded RNA-specific endoribonucleases that are essential for the maturation and decay of coding and noncoding RNAs in both prokaryotes and eukaryotes [1]
We showed that binding of RNA and DNA G-quadruplexes by Human Dicer (hDicer) precludes pre-miRNA cleavage, which suggests the existence of yet another mechanism regulating Dicer activity and miRNA biogenesis in vivo
Since the structure adopted by RNA may depend on the RNA concentration, each 12-nt RNA was assayed by polyacrylamide gel electrophoresis (PAGE), under nondenaturing conditions, at four different concentrations (0.01, 0.1, 1, and 10 μM)
Summary
Dicer belongs to the ribonuclease III (RNase III) family of double-stranded RNA (dsRNA)-specific endoribonucleases that are essential for the maturation and decay of coding and noncoding RNAs in both prokaryotes and eukaryotes [1]. Human Dicer (hDicer), consisting of 1992 amino acids (220 kDa), is one of the most structurally complex members of the RNase III family It comprises an amino (N)-terminal putative helicase domain, a domain of unknown function (DUF283), Platform, Piwi–Argonaute–Zwille (PAZ) domain, a Connector helix, two RNase III domains (RNase IIIa and RNase IIIb), and a dsRNAbinding domain (dsRBD) [2, 3]. The DUF283 domain has been implicated in the binding of single-stranded nucleic acids [5], and may be involved in interactions with the apical loop of pre-miRNA hairpins [6]. The C-terminal dsRBD is presumed to play an auxiliary role in RNA binding [8]
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