Intracellular and intercellular transport of RNA organelles in CXG repeat disorders: The strength of weak ties

RNA is a vital biomolecule, the function of which is tightly spatiotemporally regulated. RNA organelles are biological structures that either membrane-less or surrounded by membrane. They are produced by the all the cells and indulge in vital cellular mechanisms. They include the intracellular RNA granules and the extracellular exosomes. RNA granules play an essential role in intracellular regulation of RNA localization, stability and translation. Aberrant regulation of RNA is connected to disease development. For example, in microsatellite diseases such as CXG repeat expansion disorders, the mutant CXG repeat RNA’s localization and function are affected. RNA is not only transported intracellularly but can also be transported between cells via exosomes. The loading of the exosomes is regulated by RNA-protein complexes, and recent studies show that cytosolic RNA granules and exosomes share common content. Intracellular RNA granules and exosome loading may therefore be related. Exosomes can also transfer pathogenic molecules of CXG diseases from cell to cell, thereby driving disease progression. Both intracellular RNA granules and extracellular RNA vesicles may serve as a source for diagnostic and treatment strategies. In therapeutic approaches, pharmaceutical agents may be loaded into exosomes which then transport them to the desired cells/tissues. This is a promising target specific treatment strategy with few side effects. With respect to diagnostics, disease-specific content of exosomes, e.g., RNA-signatures, can serve as attractive biomarker of central nervous system diseases detecting early physiological disturbances, even before symptoms of neurodegeneration appear and irreparable damage to the nervous system occurs. In this review, we summarize the known function of cytoplasmic RNA granules and extracellular vesicles, as well as their role and dysfunction in CXG repeat expansion disorders. We also provide a summary of established protocols for the isolation and characterization of both cytoplasmic and extracellular RNA organelles

Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

Background Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. Principal Findings We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. Conclusions The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations

Charakterisierung der Mikrotubulus-assoziierten PP2A und ihrer Zielproteine

In der vorliegenden Arbeit sollten Ziel-Proteine der Mikrotubulus-assoziierten PP2A gefunden werden. Anhand phänotypischer Ähnlichkeiten zwischen OS- und Greig-, Acrocallosal- bzw. Pallister-Hall-Syndrom-Patienten wurde eine mögliche Interaktion zwischen dem MID1-alpha4-PP2A-Komplex und GLI3, einem zentralen Transkriptionsfaktor der SHH-Signaltransduktionskaskade, postuliert. In einer Reihe von zellbiologischen und proteinbiochemischen Experimenten konnte gezeigt werden, dass sowohl die intrazelluläre Lokalisation des GLI3 als auch der Phosphorylierungsstatus von Fu, einem Interaktionspartner von GLI3, über den MID1-alpha4-PP2A-Komplex und Mikrotubulus-assoziierter PP2A-Aktivität reguliert werden. Erhöhte Aktivität der Mikrotubulus-assoziierten PP2A führt hierbei zur Dephosphorylierung von Fu und zu einer Akkumulation des GLI3 im Cytosol, während verringerte PP2A-Aktivität zu einer Anreicherung der hyperphosphorylierten Form des Fu und zur Akkumulation des GLI3 im Nukleus führt. Darüber hinaus konnte GSK3beta als die der Mikrotubulus-assoziierten PP2A entgegenwirkende Kinase identifiziert werden. Eine verringerte Aktivität der GSK3beta führt zur Dephosphorylierung von Fu und zu einer Akkumulation des GLI3 im Cytosol. Außerdem wurde in der vorliegenden Arbeit eine Interaktion zwischen GLI3 und der hyperphosphorylierten Form des Fu beschrieben. Die Hyperphosphorylierung von Fu wird über die gegenläufigen Aktivitäten der Mikrotubulus-assoziierten PP2A und GSK3beta reguliert. Durch die Interaktion des hyperphosphorylierten Fu mit cytosolischem, nicht phosphorylierten GLI3 wird dessen Phosphorylierung gesteuert. Phosphoryliertes GLI3 reichert sich im Zellkern an und die Transkription von SHH-Zielgenen wird induziert. Die in dieser Arbeit identifizierten Mechanismen sind ein möglicher zellbiologischer Hintergrund der Übereinstimmung in den klinischen Erscheinungsbildern von OS und Syndromen, die mit Genen der SHH-Signaltransduktionskaskade assoziiert sind.Misregulation of microtubule-associated phosphatase 2A (PP2A) activity as a result of mutations in the ubiquitin ligase MID1 plays a central role in the pathogenesis of Opitz BBB/G syndrome (OS). Features typical for OS are shared by patients with mutations in GLI3 and PATCHED1 (PTC1), two members of the Sonic Hedgehog (SHH) pathway. These observations suggest that MID1 / PP2A may also be involved in the transduction of the SHH signal. Here we demonstrate that nuclear translocation of the transcription factor GLI3, a major effector of the SHH pathway, is regulated by the activity of the microtubule-associated pool of PP2A. This effect is reproduced pharmacologically by lithium chloride (LiCl), a potent inhibitor of glycogen synthase kinase 3beta (GSK3beta), and correlates with the phosphorylation status of human Fused (hFu), a GLI3 interaction partner. Our data suggest an antagonistic relationship between PP2A and GSK3beta as regulators of SHH signaling and provide a molecular basis for the phenotypic overlap between patients with OS and SHH pathway mutations

Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding

Aberrant RNA–protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA–protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy

Low Molecular Weight Inhibitors Targeting the RNA-Binding Protein HuR

The RNA-binding protein human antigen R (HuR) regulates stability, translation, and nucleus-to-cytoplasm shuttling of its target mRNAs. This protein has been progressively recognized as a relevant therapeutic target for several pathologies, like cancer, neurodegeneration, as well as inflammation. Inhibitors of mRNA binding to HuR might thus be beneficial against a variety of diseases. Here, we present the rational identification of structurally novel HuR inhibitors. In particular, by combining chemoinformatic approaches, high-throughput virtual screening, and RNA–protein pulldown assays, we demonstrate that the 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)benzoate ligand exhibits a dose-dependent HuR inhibition effect in binding experiments. Importantly, the chemical scaffold is new with respect to the currently known HuR inhibitors, opening up a new avenue for the design of pharmaceutical agents targeting this important protein

Targeting of endogenous MID1 by miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p leads to a reduction of AR protein.

<p>SH-SY5Y were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or MID1-specific siRNAs as positive control or a non-specific control siRNA as negative control. Upper panel: Left: MID1 as well as AR protein levels were analyzed on a western blot using specific antibodies. Actin was detected on the same membranes as loading control. A representative blot of n = 3 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p