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Daniel St Johnston - One of the best experts on this subject based on the ideXlab platform.
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Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation
The EMBO journal, 2000Co-Authors: David Micklem, Jan Adams, Stefan Grünert, Daniel St JohnstonAbstract:Drosophila Staufen Protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen Protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport.
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Miranda mediates asymmetric Protein and RNA localization in the developing nervous system.
Genes & development, 1998Co-Authors: Alison J. Schuldt, Daniel St Johnston, Jan Adams, Catherine M. Davidson, David Micklem, Jim Haseloff, Andrea H. BrandAbstract:Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda Protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding Protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3* UTR. Staufen Protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the Protein, which interacts in a yeast two-hybrid screen with Miranda Protein. Furthermore, Miranda colocalizes with Staufen Protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero Protein, also localizes prospero RNA through its interaction with Staufen Protein.
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Staufen Protein associates with the 3 utr of bicoid mrna to form particles that move in a microtubule dependent manner
Cell, 1994Co-Authors: Dominique Ferrandon, Lisa Elphick, Christiane Nussleinvolhard, Daniel St JohnstonAbstract:Abstract Staufen Protein is required in order to anchor bicoid ( bcd ) mRNA at the anterior pole of the Drosophila egg. Here we show that Staufen Protein colocalizes with bcd mRNA at the anterior, and that this localization depends upon its association with the mRNA. Upon injection into the embryo, bcd transcripts specifically interact with Staufen, and we have mapped the sequences required to three regions of the 3′UTR, each of which is predicted to form a long stem-loop. The resulting Staufen- bcd 3′UTR complexes form particles that show a microtubule-dependent localization. Since Staufen is also transported with oskar (osk) mRNA during oogenesis, Staufen associates specifically with both osk and bcd mRNAs to mediate their localizations, but at two distinct stages of development.
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Staufen, a gene required to localize maternal RNAs in the Drosophila egg
Cell, 1991Co-Authors: Daniel St Johnston, D. Beuchle, Christiane Nüsslein-volhardAbstract:The posterior group gene Staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of Staufen and show that Staufen Protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, Staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, Staufen Protein is also concentrated at the anterior pole, in the same region as bicoid RNA.
Gabriele Varani - One of the best experts on this subject based on the ideXlab platform.
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Molecular Dynamics Simulation of the RNA Complex of a Double-Stranded RNA-Binding Domain Reveals Dynamic Features of the Intermolecular Interface and Its Hydration
Biophysical journal, 2002Co-Authors: Tiziana Castrignanò, Gabriele Varani, Giovanni Chillemi, Alessandro DesideriAbstract:The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen Protein represents a paradigm to understand how the dsRBD Protein family, one of the most common RNA-binding Protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen Protein and of the separate Protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in Protein-nucleic acid interactions.
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Determination of the structure of the RNA complex of a double-stranded RNA-binding domain from Drosophila Staufen Protein
Biopolymers, 1999Co-Authors: Andres Ramos, Peter Bayer, Gabriele VaraniAbstract:We have determined using NMR the structure of the complex between the third double-stranded RNA-binding domain (dsRBD3) of Drosophila Staufen Protein and a RNA stem-loop with optimal binding properties in vitro. This work was designed to understand how dsRBD Proteins bind RNA and to investigate the role of Staufen dsRBDs in the localization of maternal RNAs during early embryonic development. The structure determination was challenging, because of weak, nonsequence specific binding and residual conformational flexibility at the RNA–Protein interface. In order to overcome the problems originated by the weak interaction, we used both new and more traditional approaches to obtain distance and orientation information for the Protein and RNA components of the complex. The resulting structure allowed the verification of aspects of RNA recognition by dsRBDs matching the information obtained by a related crystallographic study. We were also able to generate new observations that are likely to be relevant to dsRBD–RNA binding and to the physiological role of Staufen Protein.©2001 John Wiley & Sons, Inc. Biopoly( Nucleic Acid Sci) 52: 181–196, 1999/2000
Juan Ortin - One of the best experts on this subject based on the ideXlab platform.
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Interaction of influenza virus NS1 Protein and the human homologue of Staufen in vivo and in vitro.
Nucleic acids research, 1999Co-Authors: Ana Falcón, Rosa M Marion, Puri Fortes, Ana Beloso, Juan OrtinAbstract:A screening for human Proteins capable of interacting with influenza virus NS1 has been carried out using the two-hybrid genetic trap in yeast. A cDNA corresponding to the human homologue of Drosophila melanogaster Staufen Protein (hStaufen) was isolated that fulfilled all genetic controls of the two-hybrid protocol. Using a hStaufen cDNA isolated from a lambda human library, the interaction of hStaufen and NS1 Proteins was characterised in vivo and in vitro. Co-transfection of NS1 cDNA and a partial cDNA of hStaufen led to the relocalisation of recombinant hStaufen Protein from its normal accumulation site in the cytoplasm to the nuclear location of NS1 Protein. NS1 and hStaufen Proteins could be co-immunoprecipitated from extracts of co-transfected cells and from mixtures of extracts containing either Protein, as well as from extracts of influenza virus-infected cells. Furthermore, both Proteins co-localised in the ribosomal and polysomal fractions of influenza virus-infected cells. The interaction was also detected in pull-down experiments using a resin containing purified hStaufen and NS1 Protein translated in vitro. Deletion mapping of the NS1 gene indicated that a mutant Protein containing the N-terminal 81 amino acids is unable to interact with hStaufen, in spite of retaining full RNA-binding capacity. These results are discussed in relation to the possible mechanisms of action of hStaufen and its relevance for influenza virus infection.
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a human sequence homologue of Staufen is an rna binding Protein that is associated with polysomes and localizes to the rough endoplasmic reticulum
Molecular and Cellular Biology, 1999Co-Authors: Rosa M Marion, Puri Fortes, Ana Beloso, Carlos G Dotti, Juan OrtinAbstract:In the course of a two-hybrid screen with the NS1 Protein of influenza virus, a human clone capable of coding for a Protein with high homology to the Staufen Protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a λ cDNA library. The encoded Protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant Protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparent Kd of 10−9 M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like Protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like Protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like Protein associates with polysomes. These results are discussed in relation to the possible functions of the Protein.
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The mammalian Staufen Protein localizes to the somatodendritic domain of cultured hippocampal neurons: Implications for its involvement in mRNA transport
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999Co-Authors: Michael A. Kiebler, Rosa M Marion, Puri Fortes, Juan Ortin, Indradeo Hemraj, Paul Verkade, Martin Köhrmann, Carlos G DottiAbstract:In hippocampal neurons, certain mRNAs have been found in dendrites ([Steward, 1997][1]), and their localization and translation have been implicated in synaptic plasticity ([Martin et al., 1997][2]). One attractive candidate to achieve transport of mRNAs into dendrites is Staufen (Stau), a double-stranded RNA-binding Protein, which plays a pivotal role in mRNA transport, localization, and translation in Drosophila ([St. Johnston, 1995][3]). Using antibodies raised against a peptide located in the RNA-binding domain IIa and a polyclonal antibody raised against a recently cloned human Staufen homolog, we identify a 65 kDa rat homolog in cultured rat hippocampal neurons. In agreement with the exclusive somatodendritic localization of mRNAs in these cells, we find that Staufen is restricted to the same domain. By immunoelectron microscopy, we show enrichment of the mammalian homolog of Stau (mStau) in the vicinity of smooth endoplasmic reticulum and microtubules near synaptic contacts. Finally, the association of the mStau with neuronal mRNAs is suggested by the colocalization with ribonucleoProtein particles specifically in distal dendrites known to contain mRNA, ribosomes, and translation factors ([Knowles et al., 1996][4]). These results suggest a role for mStau in the polarized transport and localization of mRNAs in mammalian neurons. [1]: #ref-28 [2]: #ref-24 [3]: #ref-27 [4]: #ref-19
Yuh Nung Jan - One of the best experts on this subject based on the ideXlab platform.
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Staufen: a common component of mRNA transport in oocytes and neurons?
Trends in cell biology, 2000Co-Authors: Fabrice Roegiers, Yuh Nung JanAbstract:Mammalian homologues of Staufen, a Protein involved in localizing mRNAs during oogenesis and early central nervous system development in Drosophila, have been identified recently. The mammalian Staufen gene encodes a Protein containing several conserved double-stranded mRNA-binding domains and is expressed in hippocampal neurons. The mammalian Staufen Protein forms granules that are transported to the distal dendrite during neuronal maturation. The Staufen granules colocalize with ribonuclear particles that transport mRNA to the dendrites. These findings might provide clues to a mechanism of mRNA transport conserved in mammalian neurons and Drosophila oogenesis.
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Transient posterior localization of a kinesin fusion Protein reflects anteroposterior polarity of the Drosophila oocyte
Current biology : CB, 1994Co-Authors: Ira E. Clark, Edward Giniger, Hannele Ruohola-baker, Lily Yeh Jan, Yuh Nung JanAbstract:Abstract Background: During oogenesis in Drosophila, determinants that will dictate abdomen and germline formation are localized to the ‘polar plasm' in the posterior of the oocyte. Assembly of the polar plasm involves the sequential localization of several messenger RNAs and Proteins to the posterior of the oocyte, beginning with the localization of oskar mRNA and Staufen Protein during stages 8 and 9 of oogenesis. The mechanism by which these two early components accumulate at the posterior is not known. We have investigated whether directed transport along microtubules could be used to accomplish this localization. Results We have made a fusion Protein composed of the bacterial β -galactosidase enzyme as a reporter, joined to part of the plus-end-directed microtubule motor, kinesin, and have found that the fusion Protein transiently localizes to the posterior of the oocyte during stages 8 and 9 of oogenesis. Treatment with the microtubule-depolymerizing agent colchicine prevents both the localization of the fusion Protein and the posterior transport of oskar mRNA and Staufen Protein. Furthermore, the fusion Protein localizes normally in oocytes mutant for either oskar and Staufen, but not in other mutants in which oskar mRNA and Staufen Protein are mislocalized. Conclusion Association with a plus-end-directed microtubule motor can promote posterior localization of a reporter Protein during oogenesis. The genetic requirements for this localization and its sensitivity to colchicine, both of which are shared with the posterior transport of oskar mRNA and Staufen Protein, suggest that similar mechanisms may function in both processes.
Luc Desgroseillers - One of the best experts on this subject based on the ideXlab platform.
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A Novel Murine Staufen Isoform Modulates the RNA Content of Staufen Complexes
Molecular and cellular biology, 2000Co-Authors: Thomas Duchaîne, Hui-jun Wang, Ming Luo, Sergey V. Steinberg, Ivan R. Nabi, Luc DesgroseillersAbstract:RNA transport and localization provide an efficient way to distribute genetic information and to allow different portions of the cell to establish their own biochemical fates (12, 20, 32, 44). Examples of this process have been described in many different organisms and cell types. RNA localization and/or localized translation are linked to different biological processes such as asymmetric cell division (7, 28, 39, 45), long-term potentiation (30), synaptic transmission (40), cell motility (23), and axis formation in oocytes (44). It is now apparent that many determinants of RNA localization are conserved among these systems. The process of mRNA localization is initiated by association of RNA with one or more RNA-binding Proteins (RBPs) through a targeting signal most commonly located in the 3′ untranslated region (3′ UTR) of the transcripts. This association results in the formation of large ribonucleoProtein complexes (RNPs) (1, 13, 24). These complexes then migrate along the cytoskeleton to their final destination, where they are anchored and translated. For the entire localization process, translation of localized mRNAs needs to be tightly regulated. Specific signals are important for repressing translation during mRNA transport and derepressing translation once RNPs are properly localized (15, 21, 22). The crucial role played by the cytoskeleton in many steps of transport, anchoring, and translation (2, 34, 41, 47) is another feature which appears to be conserved in numerous RNA localization systems. The multistep process of RNA localization is dependent on specific trans-acting Proteins. Thus, it is essential to identify and characterize these Proteins and to study their localization and regulation. Recent work has identified several classes of Proteins as components of RNP involved in mRNA transport (20). These include members of the double-stranded RBP (dsRBP) family (25, 43), homologues of the zipcode-binding Protein (10, 11, 16, 36), and members of the hnRNP family (3, 17, 33). Mammalian Staufen, a member of the dsRBP family, contains four copies of the dsRBD consensus motif (29, 46), now designated dsRBD2 to dsRBD5 for consistency with the Staufen domains in Drosophila (43). In vitro, Staufen was shown to bind dsRNA without sequence specificity (29, 46). Molecular mapping of the functional domains related the RNA-binding activity mainly to dsRBD3, with a weaker activity mapped to dsRBD4 (46). Similarly, the spacer region between dsRBD4 and dsRBD5, which resembles the tubulin-binding domain of MAP1B, was shown to bind tubulin in vitro (46). In fibroblasts, Staufen is associated with polysomes and the rough endoplasmic reticulum (RER) (29, 46), whereas in neurons, it is associated with both the RER and microtubules (19). In expression studies in neurons, Staufen was demonstrated to be a component of RNA-containing granules migrating in both anterograde and retrograde manners along the dendrites (25). This was persuasive evidence that Staufen is involved in mRNA transport in mammals. However, challenging questions remain about the precise role of each Staufen isoform; the function of the multiple domains in Staufen Protein; and the nature of its interaction with RNAs, Protein cofactors, RER, and the cytoskeleton. Recent studies in Drosophila have provided important clues about the function of Staufen. In Drosophila, Staufen is necessary for bicoid and oskar mRNA localization to the anterior and posterior poles of the oocyte, respectively (21, 42), and for prospero mRNA localization in neuroblasts (7, 27, 39). Out of the five copies of the dsRBD consensus sequences, dsRBD3 was shown to bind bicoid and prospero mRNAs in vitro (27, 43); dsRBD5 was shown to be involved in Protein-Protein interactions (39), demonstrating that Staufen is involved in both RNA-Protein and Protein-Protein interactions. Although direct binding to bicoid RNA has not yet been shown in vivo, intermolecular bicoid RNA-RNA interactions are important for recruiting Staufen in the RNP complexes (14). In contrast, Staufen directly interacts with Oskar Protein via its N-terminal domain in oocytes (5) and with Inscuteable and Miranda in neuroblasts through its C-terminal half and fifth dsRBD, respectively (27, 39). Staufen expression is also important for derepression of the localised oskar mRNA translation (5). Recently we reported the molecular cloning and characterization of human (hStau) and mouse (mStau) Staufen (46). We now report that a novel endogenous Staufen isoform (mStaui) containing a six-amino-acid insertion within its major dsRBD (dsRBD3) shows a severe reduction in its RNA-binding capacity in vitro. Our results show that this isoform, along with mStau, are components of RNA-Protein complexes and that the ratio of the two isoforms is important for the proper subcellular localization of mStaui. Finally, analyses of Staufen-containing complexes demonstrate that increasing the incorporation of mStaui drastically reduces the amount of RNA in these complexes. These results provide new insights into the mechanism of regulation of mStau function in vivo.
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Localization of a human double-stranded RNA-binding Protein gene (STAU) to band 20q13.1 by fluorescence in situ hybridization.
Genomics, 1996Co-Authors: Luc Desgroseillers, Nicole LemieuxAbstract:Asymmetric transport of mRNA within the cells is mediated by RNA-binding Proteins that form, along with the mRNAs and perhaps other small RNAs, stable ribonucleoProtein complexes. However, the nature of the Protein components of these complexes in vertebrates is still unknown. In Drosophila, genetic studies have identified a number of potential genes that are necessary for localization of mRNAs in oocytes; one of the most studied is the Staufen gene. The Staufen Protein has been shown to bind to localized mRNAs in oocytes and to be expressed in somatic cells as well. To understand the mechanism of mRNA transport in mammals and characterize its components, we recently cloned and sequenced the human Staufen homolog cDNA (HGMW-approved symbol STAU). In this paper, we show that the gene is unique in the human genome and report its chromosomal localization by fluorescence in situ hybridization. The human Staufen gene maps to chromosome 20q13.1, a region that is associated with certain genetic diseases.