The effects of the 5' pocket motif of Dicer on miRNA biogenesis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE27903 | GEO | 2011/07/13
SECONDARY ACCESSION(S): PRJNA138215
REPOSITORIES: GEO
Similar Datasets
Project description:A hallmark of RNA silencing is a class of ~22 nt RNAs which are processed from dsRNA precursor by Dicer. Accurate processing by Dicer is critical for the functionality of microRNAs (miRNAs). According to the current model, Dicer measures the length by anchoring the 3' overhang of the dsRNA terminus. Here we find that human Dicer binds to the 5' end of RNA and utilizes the 5' end as an additional reference point for cleavage site selection (5' counting rule). We further identify a novel motif (5'-pocket) in Dicer, which recognizes the 5' end of RNA. By analyzing miRNA population from 5'-pocket mutant Dicer expressing Dicer-null mES, we provide that the 5' pocket is significant for Dicer processing in vivo. Examination of small RNA profiles from Dicer-null mouse embryonic stem cells transfected with either wild-type or 5' pocket mutant Dice expression plasmids.
2011-07-12 | E-GEOD-27903 | biostudies-arrayexpress
Project description:Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.
2012-12-25 | GSE41292 | GEO
Project description:Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies. Various shRNAs were expressed in Cell and processed by the RNase III enzyme Dicer. The profiles of the siRNA products were generated by deep sequencing with or without the Ago2-IP.
2012-12-25 | E-GEOD-41292 | biostudies-arrayexpress
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
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 | GSE202530 | GEO
Project description:In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are central to sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nt small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation (RdDM) pathway. Here, we determined structure of a DCL3-pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated-A1 of the guide strand and the 1-nt 3′-overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated-end of the guide RNA and dual cleavage sites formed by the paired RNaseIII domains. These structural studies, complemented by functional data, reveal insights into the dicing principle for Dicers in general.
2021-10-14 | GSE179616 | GEO
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
Project description:Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi has different functions in eukaryotes including gene regulation, antiviral innate immunity or defense against transposable elements. In mammals, RNAi is constrained by inefficient cleavage of dsRNA by Dicer because it is adapted to produce microRNAs, a different class of small RNAs. An exception of the rule is highly active RNAi in mouse oocytes, which employs a truncated Dicer isoform (ΔHEL1). A homozygous mutation of murine Dicer to express only the truncated variant causes major dysregulation of microRNAs and perinatal lethality. Here, we report the phenotype and RNAi activity in DicerΔHEL1/wt mice, which are viable, fertile, slightly smaller, and show minimal miRNome changes. At the same time, endogenous siRNA levels are increased by an order of magnitude in DicerΔHEL1/wt mice. We show that siRNA abundance is limited by available dsRNA but not by the Protein Kinase R, an innate immunity factor shown to limit siRNA biogenesis in cultured cells. Using dsRNA expressed from a transgene, functional RNAi in vivo was successfully induced in the heart. DicerΔHEL1/wt mice thus represent a new model for researching mammalian canonical RNAi in vivo and offer an unprecedented platform for addressing earlier claims about its biological roles.
2024-04-08 | GSE259395 | GEO
Project description:Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi has different functions in eukaryotes including gene regulation, antiviral innate immunity or defense against transposable elements. In mammals, RNAi is constrained by inefficient cleavage of dsRNA by Dicer because it is adapted to produce microRNAs, a different class of small RNAs. An exception of the rule is highly active RNAi in mouse oocytes, which employs a truncated Dicer isoform (ΔHEL1). A homozygous mutation of murine Dicer to express only the truncated variant causes major dysregulation of microRNAs and perinatal lethality. Here, we report the phenotype and RNAi activity in DicerΔHEL1/wt mice, which are viable, fertile, slightly smaller, and show minimal miRNome changes. At the same time, endogenous siRNA levels are increased by an order of magnitude in DicerΔHEL1/wt mice. We show that siRNA abundance is limited by available dsRNA but not by the Protein Kinase R, an innate immunity factor shown to limit siRNA biogenesis in cultured cells. Using dsRNA expressed from a transgene, functional RNAi in vivo was successfully induced in the heart. DicerΔHEL1/wt mice thus represent a new model for researching mammalian canonical RNAi in vivo and offer an unprecedented platform for addressing earlier claims about its biological roles.
2024-04-08 | GSE242870 | GEO
Project description:Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi has different functions in eukaryotes including gene regulation, antiviral innate immunity or defense against transposable elements. In mammals, RNAi is constrained by inefficient cleavage of dsRNA by Dicer because it is adapted to produce microRNAs, a different class of small RNAs. An exception of the rule is highly active RNAi in mouse oocytes, which employs a truncated Dicer isoform (ΔHEL1). A homozygous mutation of murine Dicer to express only the truncated variant causes major dysregulation of microRNAs and perinatal lethality. Here, we report the phenotype and RNAi activity in DicerΔHEL1/wt mice, which are viable, fertile, slightly smaller, and show minimal miRNome changes. At the same time, endogenous siRNA levels are increased by an order of magnitude in DicerΔHEL1/wt mice. We show that siRNA abundance is limited by available dsRNA but not by the Protein Kinase R, an innate immunity factor shown to limit siRNA biogenesis in cultured cells. Using dsRNA expressed from a transgene, functional RNAi in vivo was successfully induced in the heart. DicerΔHEL1/wt mice thus represent a new model for researching mammalian canonical RNAi in vivo and offer an unprecedented platform for addressing earlier claims about its biological roles.
2024-04-08 | GSE242869 | GEO