Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the β1-β2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

• MeSH
• ADP-ribosylační faktor 1 chemie   genetika   MeSH
• cytoplazma chemie   genetika   MeSH
• cytoskeletální proteiny chemie   genetika   MeSH
• dvouvláknová RNA chemie   genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• proteiny vázající RNA chemie   genetika   MeSH
• stabilita RNA genetika   MeSH
• vazebná místa genetika   MeSH
• vazebný motiv pro dvoušroubovici RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADP-ribosylační faktor 1 MeSH
• cytoskeletální proteiny MeSH
• dvouvláknová RNA MeSH
• proteiny vázající RNA MeSH
• STAU1 protein, human MeSH Prohlížeč

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1⋅Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.

• Klíčová slova
• Dcr-1, Dicer, Dicer-partner proteins, Loqs-PB, Loquacious, RNase III, cryo-EM, dsRBD, isomiR, miRNA, microRNA,
• MeSH
• Drosophila genetika   MeSH
• mikro RNA * genetika   metabolismus   MeSH
• proteiny Drosophily * genetika   metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• ribonukleasa III genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• mikro RNA * MeSH
• proteiny Drosophily * MeSH
• proteiny vázající RNA MeSH
• ribonukleasa III MeSH

RNase III Dicer produces small RNAs guiding sequence-specific regulations, with important biological roles in eukaryotes. Major Dicer-dependent mechanisms are RNA interference (RNAi) and microRNA (miRNA) pathways, which employ distinct types of small RNAs. Small interfering RNAs (siRNAs) for RNAi are produced by Dicer from long double-stranded RNA (dsRNA) as a pool of different small RNAs. In contrast, miRNAs have specific sequences because they are precisely cleaved out from small hairpin precursors. Some Dicer homologs efficiently generate both, siRNAs and miRNAs, while others are adapted for biogenesis of one small RNA type. Here, we review the wealth of recent structural analyses of animal and plant Dicers, which have revealed how different domains and their adaptations contribute to substrate recognition and cleavage in different organisms and pathways. These data imply that siRNA generation was Dicer's ancestral role and that miRNA biogenesis relies on derived features. While the key element of functional divergence is a RIG-I-like helicase domain, Dicer-mediated small RNA biogenesis also documents the impressive functional versatility of the dsRNA-binding domain.

• Klíčová slova
• Dicer, dsRBD, helicase, miRNA, siRNA,
• MeSH
• dvouvláknová RNA genetika   MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• mikro RNA * genetika   metabolismus   MeSH
• ribonukleasa III * genetika   MeSH
• RNA interference MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• dvouvláknová RNA MeSH
• malá interferující RNA MeSH
• mikro RNA * MeSH
• ribonukleasa III * MeSH

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

• Klíčová slova
• DExD, Dicer, PKR, RNAi, TARBP2, cryo-EM, dsRBD, dsRNA, helicase, miRNA, mirtron,
• MeSH
• mikro RNA * genetika   metabolismus   MeSH
• myši MeSH
• ribonukleasa III * metabolismus   MeSH
• RNA interference MeSH
• savci metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA * MeSH
• ribonukleasa III * MeSH
• transportní proteiny MeSH

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

• Klíčová slova
• ADAR RNA editing, Adar mutant, Aicardi-Goutieres Syndrome (AGS), CP: Molecular biology, Mavs, dsRNA, dsRNA-binding protein, innate immune sensors, protein kinase R,
• MeSH
• adenosindeaminasa * metabolismus   genetika   MeSH
• aktivace enzymů MeSH
• buňky A549 MeSH
• dvouvláknová RNA * metabolismus   MeSH
• kinasa eIF-2 * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• proteinové domény MeSH
• proteiny vázající RNA * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADAR protein, human MeSH Prohlížeč
• adenosindeaminasa * MeSH
• dvouvláknová RNA * MeSH
• kinasa eIF-2 * MeSH
• proteiny vázající RNA * MeSH

The RNA editing enzyme ADAR2 is essential for the recoding of brain transcripts. Impaired ADAR2 editing leads to early-onset epilepsy and premature death in a mouse model. Here, we report bi-allelic variants in ADARB1, the gene encoding ADAR2, in four unrelated individuals with microcephaly, intellectual disability, and epilepsy. In one individual, a homozygous variant in one of the double-stranded RNA-binding domains (dsRBDs) was identified. In the others, variants were situated in or around the deaminase domain. To evaluate the effects of these variants on ADAR2 enzymatic activity, we performed in vitro assays with recombinant proteins in HEK293T cells and ex vivo assays with fibroblasts derived from one of the individuals. We demonstrate that these ADAR2 variants lead to reduced editing activity on a known ADAR2 substrate. We also demonstrate that one variant leads to changes in splicing of ADARB1 transcript isoforms. These findings reinforce the importance of RNA editing in brain development and introduce ADARB1 as a genetic etiology in individuals with intellectual disability, microcephaly, and epilepsy.

• Klíčová slova
• ADAR2, RNA editing, epilepsy, intellectual disability, microcephaly, migrating focal seizures,
• MeSH
• adenosindeaminasa genetika   MeSH
• alely MeSH
• alternativní sestřih genetika   MeSH
• dítě MeSH
• genetická predispozice k nemoci genetika   MeSH
• genetická variace genetika   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• mikrocefalie genetika   MeSH
• předškolní dítě MeSH
• proteiny vázající RNA genetika   MeSH
• sestřih RNA genetika   MeSH
• záchvaty genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• ADARB1 protein, human MeSH Prohlížeč
• adenosindeaminasa MeSH
• proteiny vázající RNA MeSH