Single-molecule analysis of processive double-stranded RNA cleavage by Drosophila Dicer-2

Naganuma, Masahiro; Tadakuma, Hisashi; Tomari, Yukihide

doi:10.1038/s41467-021-24555-1

Cited by 22 publications

(24 citation statements)

References 34 publications

(76 reference statements)

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“…10a,b ). However, the dsRBDs were not observed in the density of the two-dimensional average of the initial binding state, suggesting that the dsRBDs of Loqs-PD have only an assisting role in the initial dsRNA binding of the helicase domain of Dcr-2, which is consistent with the single-molecule result from a previous study 19 .…”

Section: Discussionsupporting

confidence: 89%

“…Dcr-2 may sense the 5′-triphosphate of RNAs using a similar binding pocket to that demonstrated previously 12 , 27 . Recent single-molecule analysis 19 supports the conclusion from our structures in this study that terminal loading of dsRNA as the initial step and translocation along the dsRNA are required to activate the processive cleavage activity of Dcr-2.…”

Section: Discussionsupporting

confidence: 87%

“…Recent single-molecule analysis demonstrated that Dcr-2 translocates along the dsRNA after its helicase initially binds to the dsRNA terminus in the presence of ATP 19 . To capture the different steps during the translocation of Dcr-2–Loqs-PD along the dsRNA, we used cryo-EM to analyse the sample of Dcr-2 DDNN –Loqs-PD in complex with the dsRNA substrate under conditions with ATP and Mg 2+ .…”

Section: Structural States During Translocationmentioning

confidence: 99%

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Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Wang

Deng

et al. 2022

Nature

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Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2–Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2–Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2–Loqs-PD.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 87%

Section: Structural States During Translocationmentioning

confidence: 99%

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Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Wang

Deng

et al. 2022

Nature

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“…6h ). These structural observations are consistent with previous studies indicating that the Dicer-2 helicase domain initially recognizes both long dsRNA substrates with a blunt end and a 2-nt 3′-overhanging end in the dicing process 28 , 35 , 36 .…”

Section: Dsrna Recognition By Dicer-2supporting

confidence: 92%

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

Yamaguchi

Naganuma

Nishizawa

et al. 2022

Nature

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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.

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“…From the standpoint of miRNA biogenesis, the generation of functional novel mature miRNAs requires the transcription of certain genomic loci that are capable of producing RNA whose secondary structures are recognizable by Drosha and Pasha (DGCR8 in vertebrates) in the nucleus, and Dicer and Loquacious in the cytoplasm [ 9 , 34 , 35 , 36 , 37 , 38 ]. Therefore, the birth of novel miRNA requires a relatively simple prerequisite, which is loci in the genome that evolve to produce RNAs that fold in such a way that miRNA protein machineries can identify and process them, resulting in fully functional mature miRNAs [ 39 , 40 ].…”

Section: Resultsmentioning

confidence: 99%

Revised Annotation and Characterization of Novel Aedes albopictus miRNAs and Their Potential Functions in Dengue Virus Infection

Azlan

Yunus

Halim

et al. 2022

Biology

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The Asian tiger mosquito, Ae. albopictus, is a highly invasive species that transmits several arboviruses including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV). Although several studies have identified microRNAs (miRNAs) in Ae. albopictus, it is crucial to extend and improve current annotations with both the newly improved genome assembly and the increased number of small RNA-sequencing data. We combined our high-depth sequence data and 26 public datasets to re-annotate Ae. albopictus miRNAs and found a total of 72 novel mature miRNAs. We discovered that the expression of novel miRNAs was lower than known miRNAs. Furthermore, compared to known miRNAs, novel miRNAs are prone to expression in a stage-specific manner. Upon DENV infection, a total of 44 novel miRNAs were differentially expressed, and target prediction analysis revealed that miRNA-target genes were involved in lipid metabolism and protein processing in endoplasmic reticulum. Taken together, the miRNA annotation profile provided here is the most comprehensive to date. We believed that this would facilitate future research in understanding virus–host interactions, particularly in the role of miRNAs.

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Single-molecule analysis of processive double-stranded RNA cleavage by Drosophila Dicer-2

Cited by 22 publications

References 34 publications

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

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

Revised Annotation and Characterization of Novel Aedes albopictus miRNAs and Their Potential Functions in Dengue Virus Infection

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