The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.     

Alternative splicing of Staufen2 creates the nuclear export signal for CRM1 (Exportin 1)

Mammalian Staufen2 (Stau2), a brain-specific double-stranded RNA-binding protein, is involved in the localization of mRNA in neurons. To gain insights into the function of Stau2, the subcellular localization of Stau2 isoforms fused to the green fluorescence protein was examined. Fluorescence microscopic analysis showed that Stau2 functions as a nucleocytoplasmic shuttle protein. The nuclear export of the 62-kDa isoform of Stau2 (Stau2(62)) is mediated by the double-stranded RNA-binding domain 3 (RBD3) because a mutation to RBD3 led to nuclear accumulation. On the other hand, the shorter isoform of Stau2, Stau2(59), is exported from the nucleus by two distinct pathways, one of which is RBD3-mediated and the other of which is CRM1 (exportin 1)-dependent. The nuclear export signal recognized by CRM1 was found to be located in the N-terminal region of Stau2(59). These results suggest that Stau2 may carry a variety of RNAs out of the nucleus, using the two export pathways. The present study addresses the issue of why plural Stau2 isoforms are expressed in neurons.     

Measurement of dendritic mRNA transport using ribosomal markers

mRNA is transported to the dendritic regions by forming RNA granules, an aggregate of mRNA, ribosomal proteins, rRNA, and RNA-binding proteins such as Staufen. In this study, the dendritic transport of RNA granules was measured using the individual antibodies to ribosome-specific markers such as ribosomal L4 or S6 protein, and Y10B, a monoclonal antibody specific to rRNA. All the markers showed significant immunoreactivity in the dendritic regions of the hippocampal neurons. In addition, a GFP-tagged Staufen, a marker protein of the RNA granules, was colocalized with the Y10B and S6 signals in the dendrites. The S6 signals were also colocalized with the Y10B signals in the dendrites. Consistent with previous studies, the depolarization induced by KCl stimulation increased the ribosomal level, revealed by the S6 or Y10B immunostaining in the distal dendrites. These results demonstrate the utility of ribosomal markers for detecting the RNA granules or mRNA transport in dendrites.     

Role of mitogen-activated protein kinase (MAPK) docking sites on Staufen2 protein in dendritic mRNA transport

Although transport and subsequent translation of dendritic mRNA play an important role in neuronal synaptic plasticity, the underlying mechanisms for modulating dendritic mRNA transport are almost completely unknown. In this study, we identified and characterized an interaction between Staufen2 and mitogen-activated protein kinase (MAPK) with co-immunoprecipitation assays. Staufen2 utilized a docking (D) site to interact with ERK1/2; deleting the D-site decreased colocalization of Staufen2 with immunoreactive ERK1/2 in the cell body regions of cultured hippocampal neurons, and it reduced the amount of Staufen2-containing RNP complexes in the distal dendrites. In addition, the deletion completely abolished the depolarization-induced increase of Staufen2-containing RNP complexes. These results suggest that the MAPK pathway could modulate dendritic mRNA transport through its interaction with Staufen2.     

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal Neurons

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules 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, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway 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.     

The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export

The mammalian double-stranded RNA-binding proteins Staufen (Stau1 and Stau2) are involved in RNA localization in polarized neurons. In contrast to the more ubiquitously expressed Stau1, Stau2 is mainly expressed in the nervous system. In Drosophila, the third double-stranded RNA-binding domain (RBD3) of Staufen is essential for RNA interaction. When conserved amino acids within the RBD3 of Stau2 were mutated to render Stau2 defective for RNA binding, the mutant Stau2 proteins accumulate predominantly in the nucleolus. This is in contrast to wild type Stau2 that mostly localizes in the cytosol. The nuclear import is dependent on a nuclear localization signal in close proximity to the RBD3. The nuclear export of Stau2 is not dependent on CRM1 but rather on Exportin-5. We show that Exportin-5 interacts with the RBD3 of wild type Stau2 in an RNA-dependent manner in vitro but not with mutant Stau2. When Exportin-5 is down-regulated by RNA interference, only the largest isoform of Stau2 (Stau2(62)) preferentially accumulates in the nucleolus. It is tempting to speculate that Stau2(62) binds RNA in the nucleus and assembles into ribonucleoparticles, which are then exported via the Exportin-5 pathway to their final destination.     

Staufen: a common component of mRNA transport in oocytes and neurons?

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.     

Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.     

Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction

Staufen is an RNA-binding protein, first identified for its role in oogenesis and CNS development in Drosophila. Two mammalian homologs of Staufen have been identified and shown to bind double-stranded RNA and tubulin, and to function in the somatodendritic transport of mRNA in neurons. Here, we examined whether Staufen proteins are expressed in skeletal muscle in relation to the neuromuscular junction. Immunofluorescence experiments revealed that Staufen1 (Stau1) and Staufen2 (Stau2) accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers as well as at newly formed ectopic synapses. Western blot analyses showed that the levels of Stau1 and Stau2 are greater in slow muscles than in fast-twitch muscles. Muscle denervation induced a significant increase in the expression of Stau1 and Stau2 in the extrasynaptic compartment of both fast and slow muscles. Consistent with these observations, we also demonstrated that expression of Stau1 and Stau2 is increased during myogenic differentiation and that treatment of myotubes with agrin and neuregulin induces a further increase in the expression of both Staufen proteins. We propose that Stau1 and Stau2 are key components of the postsynaptic apparatus in muscle, and that they contribute to the maturation and plasticity of the neuromuscular junction.     

A novel murine Staufen isoform modulates the RNA content of Staufen complexes

Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStau(i)) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStau(i) exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStau(i) was found in large cytoplasmic granules. Markers of the RER colocalized with mStau(i)-containing granules, showing that overexpressed mStau(i) could still be associated with the RER. Cotransfection of mStau(i) with mStau relocalized overexpressed mStau(i) to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStau(i) drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.     

RNA Granule Protein 140 (RNG140), a Paralog of RNG105 Localized to Distinct RNA Granules in Neuronal Dendrites in the Adult Vertebrate Brain

RNA granules mediate the transport and local translation of their mRNA cargoes, which regulate cellular processes such as stress response and neuronal synaptic plasticity. RNA granules contain specific RNA-binding proteins, including RNA granule protein 105 (RNG105), which is likely to participate in the transport and translation of mRNAs. In the present report, an RNG105 paralog, RNG140 is described. A homolog of RNG105/RNG140 is found in insects, echinoderms, and urochordates, whereas vertebrates have both of the two genes. RNG140 and RNG105 are similar in that both bind to mRNAs and inhibit translation in vitro, induce the formation of RNA granules, are most highly expressed in the brain, and are localized to dendritic RNA granules, part of which are accumulated at postsynapses. However, they differ in several characteristics; RNG105 is highly expressed in embryonic brains, whereas RNG140 is highly expressed in adult brains. Furthermore, the granules where RNG105 or RNG140 is localized are distinct RNA granules in both cultured cells and neuronal dendrites. Thus, RNG140 is an RNA-binding protein that shows different expression and localization patterns from RNG105. Knockdown experiments in cultured neurons also are performed, which demonstrate that suppression of RNG140 or RNG105 reduces dendrite length and spine density. Knockdown effects of RNG140 were not rescued by RNG105, and vise versa, suggesting distinct roles of RNG105 and RNG140. These results suggest that RNG140 has roles in the maintenance of the dendritic structure in the adult vertebrate brain through localizing to a kind of RNA granules that are distinct from RNG105-containing granules.     

Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers

Messenger RNA localization involves the assembly of ribonucleoprotein particles (RNPs) and their subsequent transport along the cytoskeleton to their final destination. Here, we provide new evidence that microtubule-associated protein 2 (MAP2), calcium/calmodulin-dependent protein kinase II (CaMKIIα) and β-actin RNAs localize to dendrites in distinct RNPs, which contain--unexpectedly--very few RNA molecules. The number of MAP2 molecules per particle is affected by synaptic activity and Staufen 2, indicating that RNP composition is tightly controlled. Our data suggest that the independent localization of individual RNAs in low copy numbers could contribute to tighter temporal and spatial control of expression in neurons and synapse-specific plasticity.     

Nuclear export factor family protein participates in cytoplasmic mRNA trafficking

In eukaryotes, the nuclear export of mRNA is mediated by nuclear export factor 1 (NXF1) receptors. Metazoans encode additional NXF1-related proteins of unknown function, which share homology and domain organization with NXF1. Some mammalian NXF1-related genes are expressed preferentially in the brain and are thought to participate in neuronal mRNA metabolism. To address the roles of NXF1-related factors, we studied the two mouse NXF1 homologues, mNXF2 and mNXF7. In neuronal cells, mNXF2, but not mNXF7, exhibited mRNA export activity similar to that of Tip-associated protein/NXF1. Surprisingly, mNXF7 incorporated into mobile particles in the neurites that contained poly(A) and ribosomal RNA and colocalized with Staufen1-containing transport granules, indicating a role in neuronal mRNA trafficking. Yeast two-hybrid interaction, coimmunoprecipitation, and in vitro binding studies showed that NXF proteins bound to brain-specific microtubule-associated proteins (MAP) such as MAP1B and the WD repeat protein Unrip. Both in vitro and in vivo, MAP1B also bound to NXF export cofactor U2AF as well as to Staufen1 and Unrip. These findings revealed a network of interactions likely coupling the export and cytoplasmic trafficking of mRNA. We propose a model in which MAP1B tethers the NXF-associated mRNA to microtubules and facilitates their translocation along dendrites while Unrip provides a scaffold for the assembly of these transport intermediates.     

The RNA-binding protein Staufen from rat brain interacts with protein phosphatase-1

In mammalian neurones, homologues of the Drosophila RNA-binding protein Staufen are part of ribonucleoprotein complexes that move bidirectionally along dendritic microtubules and appear to regulate mRNA translocation and translation. In this study, putative components of Staufen granules were identified in a yeast two-hybrid screen of a rat brain cDNA library with a rat Staufen bait. Protein phosphatase-1 was found as an interacting partner. Binding appears to be mediated by a five amino acid residue sequence motif (R-K-V-T-F) in Staufen that is conserved in a number of proteins interacting with the phosphatase. A two amino acid residue mutation within this motif (R-K-V-G-A) disrupted the interaction. A cytoplasmic interaction of both proteins was shown by coimmunoprecipitation of rat Staufen and protein phosphatase-1 from the cytoplasm of transfected cells and rat brain homogenates. In mammalian brain, the phosphatase represents the first described endogenous interaction partner of Staufen. In primary hippocampal neurones, both proteins partially colocalize in somata and neuronal processes. Staufen does not modulate the in vitro protein phosphatase activity. These findings show that protein phosphatase-1 is a native component of Staufen particles. Cellular functions of Staufen may be regulated via phosphorylation or Staufen may recruite the phosphatase into specific ribonucleoprotein complexes.     

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55(Gag), viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55(Gag). Pr55(Gag) synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.     

Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).     

Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells

Staufen1 (Stau1) is an RNA-binding protein involved in transport, localization, decay, and translational control of mRNA. In neurons, it is present in cell bodies and also in RNA granules which are transported along dendrites. Dendritic mRNA localization might be involved in long-term synaptic plasticity and memory. To determine the role of Stau1 in synaptic function, we examined the effects of Stau1 down-regulation in hippocampal slice cultures using small interfering RNA (siRNA). Biolistic transfection of Stau1 siRNA resulted in selective down-regulation of Stau1 in slice cultures. Consistent with a role of Stau1 in transporting mRNAs required for synaptic plasticity, Stau1 down-regulation impaired the late form of chemically induced long-term potentiation (L-LTP) without affecting early-LTP, mGluR1/5-mediated long-term depression, or basal evoked synaptic transmission. Stau1 down-regulation decreased the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting a role in maintaining efficacy at hippocampal synapses. At the cellular level, Stau1 down-regulation shifted spine shape from regular to elongated spines, without changes in spine density. The change in spine shape could be rescued by an RNA interference-resistant Stau1 isoform. Therefore, Stau1 is important for processing and/or transporting in dendrites mRNAs that are critical in regulation of synaptic strength and maintenance of functional connectivity changes underlying hippocampus-dependent learning and memory.