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.     

Molecular characterization of MARTA1, a protein interacting with the dendritic targeting element of MAP2 mRNAs

In neurones, the somatodendritic microtubule-associated protein 2 regulates the stability of the dendritic cytoskeleton. Its extrasomatic localization appears to be a multicausal mechanism that involves dendritic mRNA trafficking, a process that depends on a dendritic targeting element in the 3' untranslated region. Two rat MAP2-RNA trans-acting proteins, MARTA1 and MARTA2, exhibit specific high-affinity binding to the dendritic targeting element. We have now affinity-purified MARTA1 from rat brain. Analysis of proteolytic peptides revealed that rat MARTA1 is the orthologue of the human RNA-binding protein KSRP. Rat MARTA1 is a 74-kDa protein that contains four putative RNA-binding domains and is 98% identical to human KSRP. Both purified rat MARTA1 and human KSRP preferentially bind to the dendritic targeting element, but do not strongly interact with other investigated regions of mRNAs encoding microtubule-associated protein 2 and alpha-tubulin. In rat brain neurones and cultured neurones derived from superior cervical ganglia, MARTA1 is primarily intranuclear, but is also present in the somatodendritic cytoplasm. Thus, MARTA1 may play a role in nucleocytoplasmic mRNA targeting.     

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.     

Determination of the structure of the RNA complex of a double-stranded RNA-binding domain from Drosophila Staufen protein

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.Copyright 2001 John Wiley & Sons, Inc.     

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation

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.     

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.     

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.     

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.     

RNA recognition by a Staufen double-stranded RNA-binding domain

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix α1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.     

RNA aptamers binding the double-stranded RNA-binding domain

Specific RNA recognition of proteins containing the double-strand RNA-binding domain (dsRBD) is essential for several biological pathways such as ADAR-mediated adenosine deamination, localization of RNAs by Staufen, or RNA cleavage by RNAse III. Structural analysis has demonstrated the lack of base-specific interactions of dsRBDs with either a perfect RNA duplex or an RNA hairpin. We therefore asked whether in vitro selections performed in parallel with individual dsRBDs could yield RNAs that are specifically recognized by the dsRBD on which they were selected . To this end, SELEX experiments were performed using either the second dsRBD of the RNA-editing enzyme ADAR1 or the second dsRBD of Xlrbpa, a homolog of TRBP that is involved in RISC formation. Several RNA families with high binding capacities for dsRBDs were isolated from either SELEX experiment, but no discrimination of these RNAs by different dsRBDs could be detected. The selected RNAs are highly structured, and binding regions map to two neighboring stem-loops that presumably form stacked helices and are interrupted by mismatches and bulges. Despite the lack of selective binding of SELEX RNAs to individual dsRBDS, selected RNAs can efficiently interfere with RNA editing in vivo.     

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.     

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.     

The dsRBP Staufen2 governs RNP assembly of neuronal Argonaute proteins

Mature microRNAs are bound by a member of the Argonaute (Ago1-4) protein family, forming the core of the RNA-induced silencing complex (RISC). Association of RISC with target mRNAs results in ribonucleoprotein (RNP) assembly involved in translational silencing or RNA degradation. Yet, the dynamics of RNP assembly and its underlying functional implications are unknown. Here, we have characterized the role of the RNA-binding protein Staufen2, a candidate Ago interactor, in RNP assembly. Staufen2 depletion resulted in the upregulation of Ago1/2 and the RISC effector proteins Ddx6 and Dcp1a. This upregulation was accompanied by the displacement of Ago1/2 from processing bodies, large RNPs implicated in RNA storage, and subsequent association of Ago2 with polysomes. In parallel, Staufen2 deficiency decreased global translation and increased dendritic branching. As the observed phenotypes can be rescued by Ago1/2 knockdown, we propose a working model in which both Staufen2 and Ago proteins depend on each other and contribute to neuronal homeostasis.     

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.     

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.