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

ABSTRACT: Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes^1,2. 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 duplexes^5,6. 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.

SUBMITTER: Su S

PROVIDER: S-EPMC9279154 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), plays major role in generating 21 base-pair (bp) siRNA duplexes from long double-stranded RNAs (dsRNAs). The ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes. Here we report the cryo-EM structures of Dcr-2/Loqs-PD in the apo state and in multiple processing states of a 50-bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and significant conformational changes of Dcr-2 during a dsRNA processing cycle. The N-terminal helicase and DUF283 domains undergo conformational change upon 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, while the interaction between DUF283 and RIIIDb domains prevents non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active center. Additional ATP-dependent conformational changes are required to form an active-dicing state and precisely cleave the dsRNA into 21-bp siRNA duplex 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.

2022-04-21 | PXD033365 | iProX

Loquacious-PD facilitates Drosophila Dicer-2 cleavage through interactions with the helicase domain and dsRNA.

Project description:Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs in Drosophila In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2's helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2.

| S-EPMC5617286 | biostudies-literature

The molecular mechanism of dsRNA processing by a bacterial Dicer.

Project description:Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

| S-EPMC6511835 | biostudies-literature

Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila.

Project description:In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2 and then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances.

| S-EPMC3089465 | biostudies-literature

Sequence determinants of dsRNA processing by DICER

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-12-16 | GSE202535 | GEO

Drosophila dicer-2 cleavage is mediated by helicase- and dsRNA termini-dependent states that are modulated by Loquacious-PD.

Project description:In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3' overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest-affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3'ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2's partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured, or frayed ends.

| S-EPMC4433149 | biostudies-literature

Modulation of MicroRNA Processing by Dicer via Its Associated dsRNA Binding Proteins.

Project description:MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).

| S-EPMC8482068 | biostudies-literature

Sequence determinants of dsRNA processing by DICER [Rescue Experiment]

Project description:RNA silencing relies on specific and efficient processing of dsRNA by DICER which produces microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, our current knowledge of DICER’s specificity is restricted to the secondary structures of its substrates: a dsRNA than 22 bp with a 2-nt 3′ overhang. We recently found evidence pointing to additional sequence-dependent determinant(s) beyond these features. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with over a million pre-miRNA variants. Our analyses revealed a highly conserved cis-acting element, termed the “GYM” motif (paired G, paired pyrimidine, and mismatched C or A) at the cleavage site, which strongly promotes processing at a specific position. We find that the C-terminal double-stranded RNA binding domain (dsRBD) of DICER recognizes the GYM motif. Mutation of the dsRBD alters pre-miRNA cleavage sites and impairs processing in a motif-dependent fashion, which in turn affects the miRNA repertoire in cells. Consistently, integrating the GYM motif into short hairpin RNA (shRNA) or Dicer substrate siRNA (DsiRNA) potentiates RNA interference.

2022-12-16 | GSE202533 | GEO

Structural and biochemical insights into the dicing mechanism of mouse Dicer: a conserved lysine is critical for dsRNA cleavage.

Project description:Dicer, an RNase III enzyme, initiates RNA interference by processing precursor dsRNAs into mature microRNAs and small-interfering RNAs. It is also involved in loading and activation of the RNA-induced silencing complex. Here, we report the crystal structures of a catalytically active fragment of mouse Dicer, containing the RNase IIIb and dsRNA binding domains, in its apo and Cd(2+)-bound forms, at 1.68- and 2.8-A resolution, respectively. Models of this structure with dsRNA reveal that a lysine residue, highly conserved in Dicer RNase IIIa and IIIb domains and in Drosha RNase IIIb domains, has the potential to participate in the phosphodiester bond cleavage reaction by stabilizing the transition state and leaving group of the scissile bond. Mutational and enzymatic assays confirm the importance of this lysine in dsRNA cleavage, suggesting that this lysine represents a conserved catalytic residue of Dicers. The structures also reveals a approximately 45-aa region within the RNase IIIb domain that harbors an alpha-helix at the N-terminal half and a flexible loop at the C-terminal half, features not present in previously reported structures of homologous RNase III domains from either bacterial RNase III enzymes or Giardia Dicer. N-terminal residues of this alpha-helix have the potential to engage in minor groove interaction with dsRNA substrates.

| S-EPMC2268147 | biostudies-literature

Sequence determinants of dsRNA processing by DICER [Massively Parallel Assay]

2022-12-16 | GSE202530 | GEO

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