staufen
In Drosophila the staufen gene encodes an RNA-binding protein that is essential for the correct localization of certain nurse cell-derived transcripts in oocytes. Although the mechanism underlying mRNA localization is unknown, mRNA-Staufen complexes have been shown to move in a microtubule-dependent manner, and it has been suggested that Staufen associates with a motor protein that generates the movement. This possibility was investigated using Notonecta glauca in which nurse cells also supply the oocytes with mRNA, but via greatly extended nutritive tubes comprised of large aggregates of parallel microtubules. Using a Staufen peptide antibody and RNA probes, a Staufen-like protein, which specifically binds double-stranded RNA, has been identified in the nutritive tubes of Notonecta. While the Staufen-like protein does not co-purify with microtubules from ovaries using standard procedures it does so under conditions of motor-entrapment, specifically in the presence of AMP-PNP. The Staufen-like protein is subsequently removed by ATP and GTP, but not ADP. Nucleotide-dependent binding to microtubules is typical of a motor-mediated interaction and the pattern of attachment and detachment of the Staufen-like protein correlates with that of a kinesin protein within the ovaries. These findings indicate that the Staufen-like RNA-binding protein attaches to, and is transported along, Notonecta ovarian microtubules by a kinesin motor (Hurst, 1999).
A double-stranded (ds)RNA-binding domain has been identified in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches have identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in Staufen and one in Xlrbpa, the minimal region that binds dsRNA was delimited. On the basis of the binding studies and computer analysis, a consensus sequence was derived that defines a 65- to 68-amino acid dsRNA-binding domain (St Johnston, 1992).
Searches with dsRNA-binding domain profiles detected two copies of the domain in each of the following: RNA helicase A, Drosophila Maleless and C. elegans ORF T20G5-11 (of unknown function). RNA helicase A is unusual in being one of the few characterised DEAD/DExH helicases that are active as monomers. Other monomeric DEAD/DExH RNA helicases (p68, NPH-II) have domains that match another RNA-binding motif, the RGG repeat. The DEAD/DExH domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the DEAD/DExH-box or by bound partners as in the eIF-4AB dimer. The presence or absence of extra RNA-binding domains should allow classification of DEAD/DExH proteins as monomeric or multimeric helicases (Gibson, 1994).
Although most RNA-binding proteins recognize a complex set of structural motifs in their RNA target, the double-stranded (ds) RNA-binding proteins are limited to interactions with double helices. Some dsRNA-binding proteins share regions of amino-acid similarity known as dsRNA-binding motifs. A Xenopus ovary cDNA expression library was screened with radiolabeled dsRNA to identify previously uncharacterized dsRNA-binding proteins. The analysis of an incomplete cDNA identified during the screen led to the discovery of two longer cDNAs of related sequence. The proteins encoded by these cDNAs each contain two dsRNA-binding motifs rich in glycine. The nucleic-acid-binding properties of a fusion protein containing the two dsRNA-binding motifs and the auxiliary domain were analyzed using a gel mobility shift assay. The fusion protein binds dsRNA possessing a variety of different sequences, and exhibits a preference for binding to dsRNA and RNA-DNA hybrids over other nucleic acids. Appropriate mRNAs, corresponding to each cDNA, were detected in polyadenylated RNA isolated from Xenopus stage VI oocytes, but translation of one of the mRNAs appears to be masked until meiotic maturation. It is concluded that dsRNA-binding motifs can be associated with auxiliary domains rich in arginine and glycine. These motifs can confer very tight binding to dsRNA. Binding can also occur to RNA-DNA hybrids, suggesting recognition of some aspect of the A-form helical structure that is adopted by both dsRNA and RNA-DNA hybrids (Bass, 1994).
The protein kinase DAI, the double-stranded RNA-activated inhibitor of translation, is an essential component of the interferon-induced cellular antiviral response. The enzyme is regulated by the binding of activator and inhibitor RNAs. DAI RNA-binding domain is located within the amino-terminal 171 residues. This domain contains two copies of an RNA-binding motif characterized by a high density of basic amino acids, by the presence of conserved residues, and by a probable alpha-helical structure. Deletion of either of the two motifs prevents the binding of dsRNA, but their relative positions can be exchanged, suggesting that they cooperate to interact with dsRNA. Clustered point mutations within the RNA-binding motifs and duplications of the individual motifs indicate that the first copy of the motif plays the more important role. Mutations that impair binding have similar effects on the binding of double-stranded RNAs of various lengths and of adenovirus VA RNAI, implying that discrimination between activator and inhibitory RNAs takes place subsequent to RNA binding (Green, 1992).
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 these genes, and the subject of many studies, is staufen. 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, the human staufen homolog cDNA (HGMW-approved symbol STAU) was cloned and sequenced. The gene is unique in the human genome. The human staufen gene maps to chromosome 20q13.1, a region that is associated with certain genetic diseases (DesGroseillers, 1996).
TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) consisting of this sequence binds TAR RNA with affinities similar to that of the entire TRBP, suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, it is shown that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovers TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment reveals that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. It is suggested that these proteins are related by virtue of sharing a common RNA-binding moiety (Gatignol, 1993).
TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase that has anti-viral and anti-proliferative effects. TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, PKR inhibits viral protein synthesis; over-expression of TRBP effectively counters this inhibition. TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role: cells that overexpress TRBP exhibit transformed phenotypes. These results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation (Benkirane, 1997).
Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-AT contains 45-kDa and 90-kDa subunits. Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel. The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen in a domain predicted to bind double-stranded RNA. RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562). Both proteins are located in the nucleus of Jurkat T-cells. Clones NF45 and NF90 with a polyhistidine fusion tag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode an NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506 (Kao, 1994).
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 lambda cDNA library. The encoded protein (hStaufen-like) contains 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 shows dsRNA binding activity in vitro, with an apparent Kd of 10(-9) M. Using a specific antibody, a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa was detected in human cells. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization is observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicates that hStaufen-like protein associates with polysomes (Marion, 1999).
Staufen (Stau) is a Drosophila double-stranded RNA (dsRNA)-binding protein involved in mRNA transport and localization. To understand the molecular mechanisms of mRNA transport in mammals, human (hStau) and mouse (mStau) staufen cDNAs were cloned. In humans, four transcripts arise by differential splicing of the Stau gene and code for two proteins with different N-terminal extremities. In vitro, hStau and mStau bind dsRNA via each of two full-length dsRNA-binding domains; they also bind tubulin via a region similar to the microtubule-binding domain of MAP-1B, suggesting that Stau cross-links cytoskeletal and RNA components. Immunofluorescent double labeling of transfected mammalian cells revealed that Stau is localized to the rough endoplasmic reticulum (RER), implicating this RNA-binding protein in mRNA targeting to the RER, perhaps via a multistep process involving microtubules. These results are the first demonstration of the association of an RNA-binding protein in addition to ribosomal proteins, with the RER, implicating this class of proteins in the transport of RNA to the RNA translation site (Wickham, 1999).
In hippocampal neurons, certain mRNAs have been found in dendrites, and their localization and translation have been implicated in synaptic plasticity. One attractive candidate to achieve transport of mRNAs into dendrites is Staufen (Stau), a double-stranded RNA-binding protein that plays a pivotal role in mRNA transport, localization, and translation in Drosophila. 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, a 65 kDa rat homolog was identified in cultured rat hippocampal neurons. In agreement with the exclusive somatodendritic localization of mRNAs in these cells, it has been found that Staufen is restricted to the same domain. By immunoelectron microscopy, enrichment of the mammalian homolog of Stau (mStau) is shown to be located 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. These results suggest a role for mStau in the polarized transport and localization of mRNAs in mammalian neurons (Kiebler, 1999).
The expression pattern of mStau during neuronal development in hippocampal neurons in culture was examined; several morphologically distinct events have been characterized leading to polarization of these cells. During early stages of development (stages 1-3), when polarization of neurites into axons and dendrites has not yet been achieved, mStau is present in all processes. alpha-Tubulin immunoreactivity of the same cell serves as a cytoskeletal marker to label all processes. The presence of mStau in all processes is still evident in stage 4 cells. However, mStau becomes excluded from some of the processes at stage 5 neurons. Given that the mStau-negative processes have an axon-like morphology (thin uniform diameter) and that mRNA transport in these cells occurs in dendrites but not axons, it appears that the mStau-positive processes are dendrites. In fully polarized hippocampal neurons, axons and dendrites can be distinguished by morphological, immunological, and functional criteria; also, numerous mRNAs specifically localize to dendrites. In mature hippocampal neurons mStau immunoreactivity is present in cell bodies, as well as in dendrites. MAP2 immunoreactivity of the same cell serves as a marker to identify dendrites. mStau is not found in axons. Also, mStau protein is not found in tau-1-labeled axons. Because mStau is expressed in immature axons but not in fully mature axons, the size of axons cannot be the factor preventing the detection of mStau. This fact was investigated using electron microscopy (EM). Staufen has been shown to be present in all neurites at earlier stages and becomes preferentially restricted to the somatodendritic region in fully mature neurons (Kiebler, 1999).
Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Staufen is a double-stranded RNA-binding protein expressed in rat hippocampal neurons, and is present in large RNA-containing granules which colocalize with microtubules in dendrites. Hippocampal neurons were transiently transfected with human Staufen-green fluorescent protein (GFP); fluorescent granules are found in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, the bidirectional movement is shown of these Staufen-GFP-labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 microm/min with a maximum velocity of 24. 3 microm/min. Moreover, the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, a novel, nonvesicular, microtubule-dependent transport pathway has been characterized involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery (Kohrmann, 1999).
RNAs are present in dendrites and may be used for local protein synthesis in response to synaptic activity. To begin to understand dendritic RNA targeting, a rat homolog of staufen, a Drosophila gene that participates in mRNA targeting during development, was cloned. In hippocampal neurons, rat staufen protein displays a microtubule-dependent somatodendritic distribution pattern that overlaps with dendritic RNAs. To determine whether r-staufen is required for dendritic RNA targeting, a mutant version was constructed containing the RNA binding domains (stau-RBD) but lacking the C-terminal portion potentially involved in dendritic targeting. Stau-RBD expression was restricted to the cell bodies and proximal dendrites. Expression of stau-RBD significantly decreases (while overexpression of wild-type r-staufen increases) the amount of dendritic mRNA. Taken together, these results suggest that the rat staufen protein plays an important role in the delivery of RNA to dendrites (Tang, 2001).
Staufen1, the mammalian homolog of Drosophila Staufen, assembles into ribonucleoprotein particles (RNPs), which are thought to transport and localize RNA into dendrites of mature hippocampal neurons. Therefore, whether components of the RNA localization complex in addition to Staufen are conserved was investigated. One candidate is the mammalian homolog of Drosophila Barentsz (Btz), which is essential for the localization of oskar mRNA to the posterior pole of the Drosophila oocyte and is a component of the oskar RNA localization complex along with Staufen. Mammalian Btz behaves like a nucleocytoplasmic shuttling protein. When expressed in the Drosophila egg chamber, mammalian Btz is still able to interact with Drosophila Staufen and reach the posterior pole in the wild-type oocyte, but does not rescue the btz mutant phenotype. Most interestingly, immunoprecipitation assays show that that Btz interacts with mammalian Staufen in an RNA-dependent manner through a conserved domain, which encompasses the region of homology to the Drosophila Btz protein and contains a novel conserved motif. One candidate for an RNA that mediates this interaction is the dendritically localized brain cytoplasmic 1 transcript (see Dendritic BC1 RNA: functional role in regulation of translation initiation). In addition, Btz and Staufen1 colocalize within particles in the cell body and, to a more variable extent, in dendrites of mature hippocampal neurons. Together, these data suggest that the mRNA transport machinery is conserved during evolution, and that mammalian Btz is an additional component of the dendritic RNPs in hippocampal neurons (Macchi, 2003).
RNA localization is a key mechanism for generating cell and developmental polarity in a wide variety of organisms. A role has been investigated for the Xenopus homolog of the double-stranded RNA-binding protein Staufen in RNA localization during oogenesis. Xenopus Staufen (XStau) is present in a ribonucleoprotein complex, and associates with both a kinesin motor protein and vegetally localized RNAs Vg1 and VegT. A functional role for XStau was revealed through expression of a dominant-negative version that blocks localization of Vg1 RNA in vivo. These results suggest a central role for XStau in RNA localization in Xenopus oocytes, and provide evidence that Staufen is a conserved link between specific mRNAs and the RNA localization machinery (Yoon, 2004).
One cause for the range of RNAs recognized by Staufen probably lies in the nature of the interaction between dsRBDs and dsRNA, which is generally non-sequence specific. Vg1 and VegT contain potentially double-stranded regions, but they are specifically bound by XStau in vivo. So the question remains as to how Staufen could interact specifically with disparate RNA targets. It is proposed that there are two classes of RNA-binding factors involved in RNA localization. One class recognizes and binds to RNA localization elements in a sequence-specific manner. Examples of such factors in Xenopus include Vg1 RNA-binding proteins hnRNP I and Vg1RBP/vera. This class of factors may be cell-type specific and act to establish a core ribonucleoprotein complex for transport. The other class of factors, such as XStau, may act not at the level of sequence-specific RNA recognition, but rather, recognize the core RNP complex and mediate the interaction with the localization machinery. In such a model, some dsRBDs would interact in a non-sequence specific manner with double-stranded regions of RNA presented on the RNP, while other dsRBDs could interact with protein components of the core RNP. Consistent with this idea, dsRBD2 and dsRBD5 of Drosophila Staufen do not bind RNA in vitro, whereas dsRBD1, dsRBD3 and dsRBD4 bind dsRNA sequence nonspecifically. Dominant-negative XStau234 is defective in interaction with hnRNP I, suggesting that XStau dsRBD1 or dsRBD5 could potentially facilitate interaction between XStau and hnRNP I. It is suggested that this interaction is in the context of an RNP, and hnRNP I and Vg1RBP/vera have been shown to associate with Vg1 and VegT RNAs in the nucleus, prior to recruitment of XStau to the cytoplasmic RNP. The observed biochemical interaction between XStau and kinesin could further suggest a role for XStau in motor recruitment, although this remains an issue for future investigation. Thus, Staufen may represent a central component of the RNA localization machinery, perhaps linking the localized RNP cargoes with the motors that move them (Yoon, 2004).
Staufen1 is a component of transported ribonucleoprotein complexes. Genetic
work in Drosophila has suggested that Staufen plays a role in the de-repression
of translation of oskar mRNA following localization. To determine whether
Staufen1 can play a similar role in mammals, translation of
transcripts was studied in the presence or in the absence of Staufen1. Translationally repressed mRNAs were generated by fusing the structured human immunodeficiency virus type 1 trans-activating response (TAR) element to the 5' end of a reporter transcript. In rabbit reticulocyte lysates and in mammalian cultured
cells, the addition of Staufen1 results in the up-regulation of reporter activity when translation is driven by the TAR-bearing RNA. In contrast, Staufen1 has no effect on translation of efficiently translated mRNAs lacking an apparent structured 5' end, suggesting that Staufen1-binding to the 5' end is required for enhanced translation. Consistently, Staufen1 RNA-binding activity is necessary for this translational effect. In addition, similar up-regulation of translation is observed when Staufen1 is tethered to the 5' end of mRNAs via other structured RNAs, the highest level of translational increase being obtained with the bona fide Staufen1-binding site of the Arf1 transcript. The expression of Staufen1 promotes polysomal loading of TAR-luciferase transcripts resulting in enhanced translation. These results support a model in which the expression of Staufen1 and its interaction with the 5' end of RNA and ribosomes facilitate translation initiation (Dugre-Brisson, 2005).
Mammalian Staufen (Stau)1 is an RNA binding protein that is thought to function in mRNA transport and translational control. Nonsense-mediated mRNA decay (NMD) degrades abnormal and natural mRNAs that terminate translation sufficiently upstream of a splicing-generated exon-exon junction. This study describes an mRNA decay mechanism that involves Stau1, the NMD factor Upf1, and a termination codon. Unlike NMD, this mechanism does not involve pre-mRNA splicing and occurs when Upf2 or Upf3X is downregulated. Stau1 binds directly to Upf1 and elicits mRNA decay when tethered downstream of a termination codon. Stau1 also interacts with the 3'-untranslated region of ADP-ribosylation factor (Arf)1 mRNA. Accordingly, downregulating either Stau1 or Upf1 increases Arf1 mRNA stability. These findings suggest that Arf1 mRNA is a natural target for Stau1-mediated decay, and data indicate that other mRNAs are also natural targets. This pathway is discussed as a means for cells to downregulate the expression of Stau1 binding transcripts (Kim, 2005).
It has been found, in two independently performed microarray analyses, that there are at least 22 cell mRNAs in addition to Arf1 mRNA that bind Stau1. This suggests that sense-mediated decay (SMD) is used by cells to coordinately regulate a battery of genes in response to changes in the cellular abundance or specific activity of Stau1, Upf1, or both. If binding is sufficiently downstream of the normal termination codon, then these mRNAs should, like Arf1 mRNA, be natural targets of SMD by a mechanism that is exon junction complex independent. For example, Stau1 also binds the 3'UTR of PAICS mRNA (and also elsewhere within PAICS mRNA), and downregulating Stau1 increases PAICS mRNA abundance 2-fold. Natural substrates for SMD could arise when a termination codon is generated by alternative splicing, which has been proposed to occur one-third of the time, provided that the termination codon resides a sufficient distance upstream of a Stau1 binding site. Notably, there may be mRNAs that are more efficiently targeted for SMD than those identified to date, i.e., that would have insufficient abundance to be detected in a microarray analysis of Stau1 bound transcripts. In fact, some of these may be among the mRNAs that increase in abundance when Upf1 is downregulated but not in accordance with rules that pertain to NMD (Kim, 2005).
Helicase UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with the finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. The remarkable balance NCC SMD and the two types of NMD are discussed in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis (Gong, 2009).
In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms. The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype. This study describes the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, this study has identified a function for Stau proteins in PGC migration (Ramasay, 2006).
Cellular messenger RNAs (mRNAs) are associated to proteins in the form of ribonucleoprotein particles. The double-stranded RNA-binding (DRB) proteins play important roles in mRNA synthesis, modification, activity and decay. Staufen is a DRB protein involved in the localized translation of specific mRNAs during Drosophila early development. The human Staufen1 (hStau1) forms RNA granules that contain translation regulation proteins as well as cytoskeleton and motor proteins to allow the movement of the granule on microtubules, but the mechanisms of hStau1-RNA recognition are still unclear. This study used a combination of affinity chromatography, RNAse-protection, deep-sequencing and bioinformatic analyses to identify mRNAs differentially associated to hStau1 or a mutant protein unable to bind RNA and, in this way, defined a collection of mRNAs specifically associated to wt hStau1. A common sequence signature consisting of two opposite-polarity Alu motifs was present in the hStau1-associated mRNAs and was shown to be sufficient for binding to hStau1 and hStau1-dependent stimulation of protein expression. These results unravel how hStau1 identifies a wide spectrum of cellular target mRNAs to control their localization, expression and fate (de Lucas, 2014).
RNA-binding proteins play crucial roles in directing RNA translation to neuronal synapses. Staufen2 (Stau2) has been implicated in both dendritic RNA localization and synaptic plasticity in mammalian neurons. This study reports the identification of functionally relevant Stau2 target mRNAs in neurons. The majority of Stau2-copurifying mRNAs expressed in the hippocampus are present in neuronal processes, further implicating Stau2 in dendritic mRNA regulation. Stau2 targets are enriched for secondary structures similar to those identified in the 3' UTRs of Drosophila Staufen targets. Stau2 was shown to regulate steady-state levels of many neuronal RNAs, and its targets are predominantly downregulated in Stau2-deficient neurons. Detailed analysis confirms that Stau2 stabilizes the expression of one synaptic signaling component, the regulator of G protein signaling 4 (Rgs4) mRNA, via its 3' UTR. This study defines the global impact of Stau2 on mRNAs in neurons, revealing a role in stabilization of the levels of synaptic targets (Heraud-Farlow, 2013).
Double-stranded RNA-binding proteins are key elements in the intracellular localization of mRNA and its local translation. Staufen is a double-stranded RNA binding protein involved in the localised translation of specific mRNAs during Drosophila early development and neuronal cell fate. The human homologue Staufen1 forms RNA-containing complexes that include proteins involved in translation and motor proteins to allow their movement within the cell, but the mechanism underlying translation repression in these complexes is poorly understood. This study shows that human Staufen1-containing complexes contain essential elements of the gene silencing apparatus, like Ago1-3 proteins, and a set is described of miRNAs specifically associated to complexes containing human Staufen1. Among these, miR-124 stands out as particularly relevant because it appears enriched in human Staufen1 complexes and is over-expressed upon differentiation of human neuroblastoma cells in vitro. In agreement with these findings, expression of human Staufen1 was shown to be essential for proper dendritic arborisation during neuroblastoma cell differentiation, yet it is not necessary for maintenance of the differentiated state, and suggest potential human Staufen1 mRNA targets involved in this process (Peredo, 2014: PubMed).
Throughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. Staufen proteins consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2)-like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. This study shows that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of these findings. In summary, these data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau-family proteins of different species (Heber, 2019).
In Drosophila melanogaster, PD isoform of the double-stranded RNA binding protein (dsRBP) Loquacious (Loqs-PD) facilitates dsRNA cleavage to siRNA by Dicer-2. StaufenC (StauC) was discovered as a coleopteran-specific dsRBP required for dsRNA processing in coleopteran insects. This study shows that StauC is essential for the high RNAi efficiency observed in coleopterans. Knockdown of StauC but not the homologs of Loqs-PD and R2D2 evoked a long-lasting insensitivity to RNAi in the coleopteran cell line, Ledp-SL1. The dsRNA insensitivity induced by StauC knockdown could not be overcome merely by an increase in dose or time of exposure to dsRNA or expression of Loquacious or R2D2. Furthermore, StauC but not Loqs and R2D2 are required for processing of dsRNA into siRNA. StauC overexpression also partly restored the impaired RNAi caused by the knockdown of Loqs-PD in D. melanogaster Kc cells. However, StauC was unable to compensate for the loss-of-the function of Dcr-2 or R2D2. Overall, these data suggest that StauC functions like Lops-PD in processing dsRNA to siRNA (Kim, 2021).
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