Monika Rehbein

Identification of a cis -Acting Dendritic Targeting Element in MAP2 mRNAs

In neurons, a limited number of mRNAs have been identified in dendritic processes, whereas other transcripts are restricted to the cell soma. Here we have investigated the molecular mechanisms underlying extrasomatic localization of mRNAs encoding microtubule-associated protein 2 (MAP2) in primary neuronal cultures. Vectors expressing recombinant mRNAs were introduced into hippocampal and sympathetic neurons using DNA transfection and microinjection protocols, respectively. Chimeric mRNAs containing the entire 3′ untranslated region of MAP2 transcripts fused to a nondendritic reporter mRNA are detected in dendrites. In contrast, RNAs containing MAP2 coding and 5′ untranslated regions or tubulin sequences are restricted to the cell soma. Moreover, 640 nucleotides from the MAP2 3′ untranslated region (UTR) are both sufficient and essential for extrasomatic localization of chimeric mRNAs in hippocampal and sympathetic neurons. Thus, a cis-acting dendritic targeting element that is effective in two distinct neuronal cell types is contained in the 3′ UTR of MAP2 transcripts. The observation of RNA granules in dendrites implies that extrasomatic transcripts seem to assemble into multimolecular complexes that may function as transport units.

Identification of a cis-acting dendritic targeting element in the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II

In mammalian neurons a selected group of mRNAs, including the transcript encoding the alpha subunit of Ca2+/calmodulin‐dependent protein kinase II, is found in dendrites. The molecular mechanisms underlying extrasomatic RNA trafficking are not well described. It is thought that dendritic transcripts contain cis‐acting elements that direct their selective subcellular sorting. Here we report the identification of an extrasomatic targeting element in the 3′ untranslated region of the mRNA encoding the alpha subunit of Ca2+/calmodulin‐dependent protein kinase II. In primary hippocampal neurons, this 1200‐nucleotide‐spanning, cis‐acting element is sufficient to mediate dendritic localization of chimeric reporter transcripts. The trafficking signal does not share any striking sequence similarity with a previously characterized dendritic targeting element in transcripts encoding the microtubule‐associated protein 2. In dendrites of transfected primary neurons, recombinant RNAs form granules with an average diameter of 0.45 µm that may represent preferential RNA docking sites or multimolecular transport units. These findings imply that extrasomatic sorting of individual dendritic mRNAs involves at least partially distinct molecular mechanisms, as well as large trafficking 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 α‐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.

Characterization of Staufen 1 ribonucleoprotein complexes

In Drosophila oocytes and neuroblasts, the double-stranded RNA binding protein Staufen assembles into ribonucleoprotein particles, which mediate cytoplasmic mRNA trafficking and translation. Two different mammalian orthologues also appear to reside in distinct RNA-containing particles. To date, relatively little is known about the molecular composition of Staufen-containing ribonucleoprotein complexes. Here, we have used a novel one-step affinity purification protocol to identify components of Staufen 1-containing particles. Whereas the nucleocytoplasmic RNA-binding protein nucleolin is linked to Staufen in an RNA-dependent manner, the association of protein phosphatase 1, the microtubule-dependent motor protein kinesin and several components of the large and small ribosomal subunits with Staufen ribonucleoprotein complexes is RNA-independent. Notably, all these components do not co-purify with a second RNA-binding protein, hnRNPK (heterogeneous ribonucleoprotein K), demonstrating the high specificity of the purification protocol. Furthermore, pull-down and immunoprecipitation experiments suggest a direct interaction between Staufen 1 and the ribosomal protein P0 in vitro as well as in cells. In cell fractionation and sucrose gradient assays, Staufen co-fractionates with intact ribosomes and polysomes, but not with the isolated 40 S ribosomal subunit. Taken together, these findings imply that, in the cytoplasm of mammalian cells, an association with the ribosomal P-stalk protein P0 recruits Staufen 1 into ribosome-containing ribonucleoprotein particles, which also contain kinesin, protein phosphatase 1 and nucleolin.

Two rat brain Staufen isoforms differentially bind RNA

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule‐associated protein 2 (MAP2). Recently, we identified a cis‐acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri‐hybrid system to identify potential trans‐acting RNA‐binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA‐binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau‐I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double‐stranded RNA‐binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2‐DTE and various additional RNA fragments in an in vitro north‐western assay, rStau‐I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

The rat insulin-degrading enzyme: Molecular cloning and characterization of tissue-specific transcripts

The primary structure of the rat insulin‐degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl‐terminal consensus sequence A/S‐K‐L for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.

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.

Synthesis of two SAPAP3 isoforms from a single mRNA is mediated via alternative translational initiation

In mammalian neurons, targeting and translation of specific mRNAs in dendrites contribute to synaptic plasticity. After nuclear export, mRNAs designated for dendritic transport are generally assumed to be translationally dormant and activity of individual synapses may locally trigger their extrasomatic translation. We show that the long, GC-rich 5′-untranslated region of dendritic SAPAP3 mRNA restricts translation initiation via a mechanism that involves an upstream open reading frame (uORF). In addition, the uORF enables the use of an alternative translation start site, permitting synthesis of two SAPAP3 isoforms from a single mRNA. While both isoforms progressively accumulate at postsynaptic densities during early rat brain development, their levels relative to each other vary in different adult rat brain areas. Thus, alternative translation initiation events appear to regulate relative expression of distinct SAPAP3 isoforms in different brain regions, which may function to influence synaptic plasticity.

The RNA-binding protein MARTA2 regulates dendritic targeting of MAP2 mRNAs in rat neurons.

Dendritic targeting of mRNAs encoding the microtubule‐associated protein 2 (MAP2) in neurons involves a cis‐acting dendritic targeting element. Two rat brain proteins, MAP2‐RNA trans‐acting protein (MARTA)1 and MARTA2, bind to the cis‐element with both high affinity and specificity. In this study, affinity‐purified MARTA2 was identified as orthologue of human far‐upstream element binding protein 3. In neurons, it resides in somatodendritic granules and dendritic spines and associates with MAP2 mRNAs. Expression of a dominant‐negative variant of MARTA2 disrupts dendritic targeting of endogenous MAP2 mRNAs, while not noticeably altering the level and subcellular distribution of polyadenylated mRNAs as a whole. Finally, MAP2 transcripts associate with the microtubule‐based motor KIF5 and inhibition of KIF5, but not cytoplasmic dynein function disrupts extrasomatic trafficking of MAP2 mRNA granules. Thus, in neurons MARTA2 appears to represent a key trans‐acting factor involved in KIF5‐mediated dendritic targeting of MAP2 mRNAs.

Extrasomatic targeting of MAP2, vasopressin and oxytocin mRNAs in mammalian neurons.

In eukaryotic cells, individual cellular subregions are functionally specialized. This is reflected by an uneven distribution of particular organelles and molecular components. Neurons possess an intrinsic polarity by which intercellular input and output sites are separated to dendrites and axons, respectively. To maintain this structural and functional polarity it is crucial for a neuron to target individual proteins to their correct subcellular destination. Extrasomatic mRNA trafficking and translation is thought to contribute to subcellular protein sorting in neurons. Different isoforms of the microtubuleassociated protein 2 (MAP2) and the respective mRNAs are both found in somata and dendrites of mammalian central nervous system neurons. MAP2 is known to influence microtubule stability and rigidity in vitro and in vivo. In neurons, MAP2 may be involved in regulating the morphology and function of dendritic spines and shafts. Extrasomatic transcript localization and local protein synthesis may provide a significant contribution to these regulatory processes. In magnocellular neurons of the hypothalamus, mRNAs encoding the vasopressin- and oxytocin-precursor protein are sorted to axons and dendrites. Both neuropeptides serve a dual function. After their secretion into the systemic circulation they act as hormones on different peripheral tissues. Moreover, they are released from dendrites into the central nervous system where they probably function as neurotransmitters/neuromodulators in events related to neuronal plasticity. In this chapter, we will focus on the recent identification of cis-acting extrasomatic targeting elements in MAP2, vasopressin and oxytocin mRNAs.