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Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA.

ABSTRACT: Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-stranded RNA (dsRNA), which in turn leads to induction of the interferon-response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units. Failure to do so has catastrophic effects on cell function.

SUBMITTER: White E

PROVIDER: S-EPMC4129937 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA.

White Eleanor E Schlackow Margarita M Kamieniarz-Gdula Kinga K Proudfoot Nick J NJ Gullerova Monika M

Nature structural & molecular biology 20140511 6

Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-str ...[more]

PMID: 24814348

Similar Datasets

Human nuclear Dicer restricts the deleterious accumulation of endogenous double strand RNA

Project description:Dicer is a central enzymatic player in RNA interference (RNAi) pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, there is little known about any possible nuclear function. Here we show that Dicer is present in both the nucleus and cytoplasm, but that its nuclear levels are tightly regulated. In its nuclear manifestation Dicer interacts with RNA polymerase II (Pol II) at actively-transcribed gene loci. Loss of Dicer causes the appearance of endogenous dsRNA, leading to induction of the interferon response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping non-coding RNA transcription units. Failure to do so has catastrophic effects on cell function. Taken together, we present here a micro (mi)RNA independent role for human nuclear Dicer. DICER ChIP-seq profile in wt 293 HEK cells, and dsRNA-seq profile in wt and DICER-depleted 293 HEK cells

2014-04-10 | E-GEOD-56648 | biostudies-arrayexpress

Human nuclear Dicer restricts the deleterious accumulation of endogenous double strand RNA

2014-04-10 | GSE56648 | GEO

Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage.

Project description:The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform-Piwi/Argonaute/Zwille-connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks. We further demonstrate that turnover of damage-induced nuclear, double-stranded (ds) RNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes endogenous DNA damage and delays the DDR by impaired recruitment of repair factors MDC1 and 53BP1. Collectively, we place Dicer within the context of the DDR by demonstrating a DNA damage-inducible phosphoswitch that causes localized processing of nuclear dsRNA by p-Dicer to promote DNA repair.

| S-EPMC5551710 | biostudies-literature

Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage

Project description:The endonuclease Dicer is a key component of the human RNA interference (RNAi) pathway and known for its role in cytoplasmic micro RNA (miRNA) production. Recent findings suggest that non-canonical Dicer generates small non-coding RNA (ncRNA) to mediate the DNA damage response (DDR). Here we show that human Dicer is phosphorylated in the platform-PAZ-connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks (DSBs). We further demonstrate that turnover of damage-induced nuclear dsRNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes spontaneous DNA damage and delays DNA repair. Taken together, we place Dicer in context of the DDR by demonstrating DNA damage-inducible phospho-switch that causes localised processing of nuclear dsRNA by p-Dicer to promote DNA repair.

2018-12-08 | GSE75892 | GEO

Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins.

Project description:Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.

| S-EPMC5716075 | biostudies-literature

In vitro binding of single-stranded RNA by human Dicer.

Project description:While Dicer alone has been shown to form stable complexes with double-stranded RNAs and short interfering RNAs, its interactions with single-stranded RNAs (ssRNAs) have not been characterized. Here, we show that recombinant human Dicer alone can bind 21-nt ssRNAs in vitro, independent of their sequence and structure. We also demonstrate that Dicer binds ssRNAs having a 5'-phosphate with greater affinity versus those with a 5'-hydroxyl. In addition, 3'-biotinylated ssRNAs are bound by Dicer with lower affinity than 3'-hydroxyl ssRNAs. The stability of ssRNA-Dicer complexes was found to depend on divalent cations. Together, our results suggest a role for the PAZ domain of Dicer in binding ssRNAs and may indicate roles for Dicer in cellular function beyond those currently known.

| S-EPMC2206536 | biostudies-literature

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

Project description:Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.

| S-EPMC8277814 | biostudies-literature

Loquacious-PD regulates the terminus-dependent molecular recognition of Dicer-2 toward double-stranded RNA.

Project description:Dicer-2 cleaves double-stranded RNA into siRNAs in a terminus-dependent manner as part of D. melanogaster's RNA interference pathway. Using ultrafast fluorescence, we probe the local environment of chromophores at the dsRNA terminus upon binding by Dicer-2 and interrogate the effects of Loquacious-PD, an accessory protein. We find substrate-selective modes of molecular recognition that distinguish between blunt and 3'overhang termini, but whose differences are greatly reduced by Loquacious-PD. These results connect the molecular recognition properties of Dicer-2 to its selective processing of dsRNAs with different termini and to its need for Loquacious-PD to efficiently produce endogenous siRNAs.

| S-EPMC8830880 | biostudies-literature

Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure.

Project description:Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3' single-stranded (3'-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3'-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.

| S-EPMC4747904 | biostudies-other

Hypoxia Regulates Endogenous Double-Stranded RNA Production via Reduced Mitochondrial DNA Transcription.

Project description:Hypoxia is a common phenomenon in solid tumours strongly linked to the hallmarks of cancer. Hypoxia promotes local immunosuppression and downregulates type I interferon (IFN) expression and signalling, which contribute to the success of many cancer therapies. Double-stranded RNA (dsRNA), transiently generated during mitochondrial transcription, endogenously activates the type I IFN pathway. We report the effects of hypoxia on the generation of mitochondrial dsRNA (mtdsRNA) in breast cancer. We found a significant decrease in dsRNA production in different cell lines under hypoxia. This effect was HIF1α/2α-independent. mtdsRNA was responsible for induction of type I IFN and significantly decreased after hypoxia. Mitochondrially encoded gene expression was downregulated and mtdsRNA bound by the dsRNA-specific J2 antibody was decreased during hypoxia. These findings reveal a new mechanism of hypoxia-induced immunosuppression that could be targeted by hypoxia-activated therapies.

| S-EPMC8651540 | biostudies-literature

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