Structure of the Dicer-2–R2D2 heterodimer bound to a small RNA duplex

Abstract

In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts1. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation2–5. Here we report cryo-electron microscopy structures of the Dicer-2–R2D2 and Dicer-2–R2D2–siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing.

Structural Insights into the Advances and Mechanistic Understanding of Human Dicer

“…These include structures of Drosophila Dicer-2, Apo-hsDicer-TRBP complex, hsDicer-TRBP bound to pre-let-7 in a pre-cleavage state, and a cleavage-competent structure of Arabidopsis Dicer-like-protein 3 (DCL3) bound to pre-siRNA . In addition to these structures, new structures continue to be reported including six Drosophila Dicer-1–Loquacious-PB (Loqs-PB) complex structures, six Drosophila Dicer-2–Loqs-PD complex structures, and two Drosophila Dicer-2–R2D2 complex structures . The 14 Drosophila Dicer structures depict dmDicer-1 and dmDicer-2 both apo- and in complex with dsRNA spanning a variety of conformational states.…”

“…50,51,75−78 Additionally, separate structural studies of dmDicer-2 bound to R2D2 in complex with siRNA revealed structural information for a strand selection/transfer conformation (Figure 4). 53 Together, these structures depict a nearly complete stepwise mechanism for dmDicer-2 ATP-dependent activity. However, while these structures may suggest that we have learned all we can about dmDicer-2, a unique dmDicer-2 dimer which is not shown in Figure 4 was also determined, revealing that, in the absence of ATP, dmDicer-2 dimerizes to form an initial binding complex with dsRNA.…”

“…The defining structural difference between Class II and Class III proteins is the addition of an N-terminal duplex RNA-activated ATPase (DRA) helicase domain, also sometimes referred to as a DExD/H-box helicase domain. − The DRA helicase domain, formed by three subdomains, Hel1, Hel2i, and Hel2, binds and unwinds the hairpin loop region of the pre-miRNA substrate. ,− As shown in Figure , while the majority of Dicer and Dicer-like enzymes contain an N-terminal DRA helicase domain, there is variability between species regarding whether this domain is ATP dependent or independent. These differences in ATP dependence are seemingly linked to Dicer’s substrate specificity during cleavage. ,− For instance, in Drosophila , Dicer-1 is ATP independent and primarily cleaves pre-miRNA, while Dicer-2 requires ATP to cleave pre-small interfering RNAs (siRNA). ,− Interestingly, the minimal Giardia Dicer lacks the helicase domain altogether despite being classified as a Class III RNase III enzyme . Lack of a helicase domain may suggest an evolutionary shift in Dicer enzymes requiring a more sophisticated means to differentiate RNA substrates in higher eukaryotes.…”

“…Figure outlines some of these structures in their proposed mechanistic sequence and compares this structural information to what is currently available in regard to hsDicer. Missing from Figure are additional structures of dmDicer-1 in complex with Loqs-PB determined in the presence of Ca 2+ , as well as dmDicer-1 bound to dsRNA in an intermediate cleavage state, a dimer structure of dmDicer-2 in complex with Loqs-PD, and apo-DmDicer-2 in complex with R2D2 …”

“…Early attempts at studying full-length hsDicer to better characterize the helicase domain and additional noncatalytic domains were done in 2009 and 2012 using negative stain transmission electron microscopy (TEM). , Although only reaching ∼20 Å resolution, the structures showed that Dicer forms a L-shaped structure and provided an initial model for the structural organization of hsDicer. Later advancements in the field of cryo-EM, both instrumentation and computation, made it possible to determine structures of full-length Dicer from humans, Drosophila , and Arabidopsis at resolutions amenable for building molecular models despite its highly flexible nature. − ,,, As a result, by the end of 2021, the first four full-length Dicer and Dicer-like structures were published with resolutions spanning 7 to 3.1 Å. These include structures of Drosophila Dicer-2, Apo-hsDicer-TRBP complex, hsDicer-TRBP bound to pre-let-7 in a pre-cleavage state, and a cleavage-competent structure of Arabidopsis Dicer-like-protein 3 (DCL3) bound to pre-siRNA .…”