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Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes \"Slicer\" activity to RISC, providing the catalytic engine for RNAi.

In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage 1 . Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies 2 , 3 . Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5′ phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

Sequencing of complete genomes has provided researchers with a wealth of information to study genome organization, genetic instability, and polymorphisms, as well as a knowledge of all potentially expressed genes. The identification of all genes encoded in the human genome opens the door for large-scale systematic gene silencing using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). With the recent development of siRNA and shRNA expression libraries, the application of RNAi technology to assign function to cancer genes and to delineate molecular pathways in which these genes affect in normal and transformed cells, will contribute significantly to the knowledge necessary to develop new and also improve existing cancer therapy.

Intranasal drug delivery is emerging as a transformative strategy for treating neurodegenerative diseases, offering a noninvasive route that bypasses the limitations of the blood–brain barrier. This review provides a comprehensive analysis of the current landscape and challenges of intranasal drug delivery for neurodegenerative conditions with an emphasis on nanoparticle (NP)‐based formulations. Unlike traditional systemic methods, intranasal delivery uses the nasal cavity’s anatomy to achieve rapid and direct central nervous system transport via the olfactory and trigeminal pathways. This review evaluates NP strategies designed to overcome physiological barriers such as mucociliary clearance, enzymatic degradation, and limited nasal volume capacity. Innovations in mucoadhesive coatings, surface modifications, and stimuli‐responsive NP that optimize drug release and retention time within the nasal cavity are highlighted. In addition,​ cutting‐edge NP systems under clinical investigation and their translational potential are explored, addressing challenges like reproducibility, safety, and patient adherence. New research directions for optimizing NP formulations to improve outcomes in diseases like Alzheimer’s, Parkinson’s, and hereditary ataxias are proposed. Lastly, broader implications of these technologies for conditions such as psychiatric disorders and brain injuries are discussed. By integrating nanotechnology and intranasal administration, this review aims to inspire future research to bridge the gap between preclinical promise and clinical success, advocating for continued innovation to unlock the full therapeutic potential of this promising approach for treating neurological disorders.

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb‐girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O ‐glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O ‐glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle‐specific α‐dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7 + cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch‐dependent loss of satellite cells. Synopsis A protein O ‐glucosyltransferase 1 ( POGLUT1 ) homozygous D233E mutation underlies a novel autosomal recessive muscular dystrophy, wherein altered Notch signaling affects muscle regeneration and, as a consequence, α‐dystroglycan glycosylation. POGLUT1 D233E exhibits decreased enzymatic activity toward Notch EGF repeats. POGLUT1 D233E leads to Notch activity downregulation, which affects muscle regeneration due to satellite cell (SC) loss of quiescence, depletion of PAX7 + cells, and premature and enhanced differentiation. Reduced Notch signaling accelerates muscle differentiation and disrupts the progressive and coordinated process of α‐dystroglycan glycosylation during differentiation, and hence, mild α‐dystroglycan hypoglycosylation is observed in skeletal muscle from POGLUT1 D233E patients. Defective regeneration, combined with α‐dystroglycan hypoglycosylation, likely results in skeletal muscle degeneration and finally gives rise to muscular dystrophy. Graphical Abstract A protein O ‐glucosyltransferase 1 ( POGLUT1 ) homozygous D233E mutation underlies a novel autosomal recessive muscular dystrophy, wherein altered Notch signaling affects muscle regeneration and, as a consequence, α‐dystroglycan glycosylation.

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb‐girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O‐glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O‐glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle‐specific α‐dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch‐dependent loss of satellite cells.