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Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs1

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Abstract

Different isoforms of the microtubule-associated protein 2 (MAP2) are somatodendritic components of neurons that seem to regulate the stability of the dendritic cytoskeleton. MAP2 localization into dendrites appears to be a complex multicausal mechanism that involves the specific recruitment of MAP2 mRNAs into dendritic compartments. Recently, we have functionally characterized a 640-nucleotide dendritic targeting element (DTE) in the 3′ untranslated region (3′ UTR) of MAP2 transcripts that mediates extrasomatic mRNA localization in primary neurons (Blichenberg et al., 1999). In analogy to molecular mechanisms regulating cytoplasmic RNA translocation in other cell systems, we propose that, in vivo, the cis-acting MAP2-DTE interacts with specific protein factors present in neurons. To identify putative trans-acting DTE-binding proteins, we performed in vitro ultraviolet crosslinking assays. Using this experimental system, two 90-kDa and 65-kDa MAP2-RNA trans-acting proteins, MARTA1 and MARTA2, were identified in rat-brain extracts. Both MARTAs bind with high affinity to the MAP2-DTE, but not to other investigated regions of MAP2 transcripts or the somatically restricted α-tubulin mRNA. Moreover, MARTA1 and MARTA2 do not bind significantly to other dendritically localized transcripts encoding vasopressin and arg3.1, nor to a dendritic trafficking element from the mRNA encoding the α-subunit of the Ca2+/calmodulin-dependent protein kinase II. Binding of MARTA1 and MARTA2 to the MAP2-DTE occurs with an affinity in the nanomolar range. Whereas MARTA1 is clearly detectable in crude lysates, cytosolic and ribosomal salt-wash fractions, and in nuclear extracts, MARTA2 is preferentially found in the ribosomal salt-wash preparation. Neither MARTA is restricted to rat brain, and both are present in a number of other rat tissues. Thus, both proteins may be involved in a variety of nuclear and cytoplasmic events that regulate RNA metabolism in different cell types.

Introduction

Cell compartmentalization is an integral feature of eukaryotic cells, allowing their segregation into distinct functional subunits. The specialized functions of individual cellular subdomains require a specific protein composition. Two basic molecular mechanisms are thought to contribute to differential protein localization patterns; a selective protein sorting, and a locally restricted, regulated translation of specific mRNAs in subcellular regions [2], [3], [4], [21], [54], [62], [68]. In neurons [33], [37], [46], [60], [66], oligodendrocytes [12], chicken myo- and fibroblasts [3], [34], [57], Drosophila embryos [15], [25], [41], [56], [62] and Xenopus oocytes [17], [20], [27], [47], [48], [61], regulated interactions of cis-acting RNA elements and trans-acting proteins seem to mediate cytoplasmic targeting and site-specific translation of numerous mRNAs. In addition, cytoskeletal components are likely to play a crucial role for transcript localization, translation and stability [3], [52].

In mammalian neurons, dendritic targeting of selected RNAs is an energy-dependent mechanism [16] that seems to require filaments of the cytoskeleton [3], [5], [30], [35], [43]. The ultrastructural identification of polysomes in dendrites spurred the idea that extrasomatic transcripts may be locally translated in dendritic processes [64], [65]. The capacity of isolated dendrites to synthesize proteins supported this concept [14], [67]. Moreover, a specific type of synaptic plasticity seems to depend on dendritic translation [31]. Taken together, these and additional data suggest that the molecular composition of individual dendritic sections may, in part, be regulated by an extrasomatic synthesis of selected proteins [33], [37], [46], [60], [66].

Whereas parts of the molecular mechanisms involved in cytoplasmic mRNA localization and local translation are well described in some non-neuronal cell systems, the molecular players mediating dendritic-transcript targeting and protein biosynthesis are poorly characterized. Recently, we defined a 640-nucleotide cis-acting dendritic targeting element (DTE) in the 3′ untranslated region (3′ UTR) of mRNAs that encode distinct isoforms of the microtubule-associated protein 2 (MAP2). In primary neurons, the MAP2-DTE mediates selective transcript localization into dendrites [8]. To describe trans-acting neuronal proteins that specifically interact with the cis-element in MAP2 mRNAs, and may thus be involved in dendritic transcript trafficking, we performed ultraviolet (UV) crosslinking assays. In adult rat-brain extracts, we identified two major trans-acting factors, MARTA1 and MARTA2, which bind to the DTE in MAP2 transcripts with an affinity in the nanomolar range. In contrast, both MARTAs interact only very weakly with sequences of the MAP2 coding region, some other dendritic transcripts and the somatically localized α-tubulin mRNA. MARTA2 is highly enriched in the ribosomal salt-wash (RSW) fraction, whereas MARTA1 is found in crude lysates, cytosolic and RSW fractions, as well as in nuclear extracts. Thus, in neurons, MARTA1 and MARTA2 may be involved in transport and translation of MAP2 transcripts.

Section snippets

Subcellular fractionation and preparation of protein extracts

Wistar rats were anaesthetized with CO2 and killed by decapitation. Organs were removed, frozen in liquid nitrogen, chopped into small pieces and homogenized on ice with a five-fold volume of extraction buffer [10 mM HEPES, pH 7.6, 3 mM MgCl2, 40 mM KCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT), 0.2% (v/v) Nonidet-P-40, protease inhibitor cocktail Complete (Roche Diagnostics, Mannheim, Germany)] in a Potter glass homogenizer with a tight-fitting Teflon pestle.

Primary glial cell cultures

Identification of MAP2-DTE interacting proteins from rat brain

Recently, we have functionally determined a cis-acting DTE in the 3′ UTR of MAP2 mRNAs that directs extrasomatic transcript localization in primary neurons [8]. To identify putative trans-acting proteins binding to the MAP2-DTE, we performed UV crosslinking assays with extracts from total rat brain. It is generally assumed that the formation of UV-induced crosslinks between proteins and RNA occurs only at sites of close contact. Therefore, proteins detected by UV crosslinking assays are likely

Discussion

Subcellular mRNA localization and translation is thought to play an important role in cell polarization [2], [3], [4], [30], [62]. In neurons, dendritic targeting of specific transcripts seems to contribute to protein compartmentalization as well as synaptic plasticity [33], [37], [46], [60], [66]. It is thought that the molecular machinery underlying mRNA trafficking in dendrites involves cis-acting signal sequences on localized transcripts that associate with trans-acting protein factors. We

Acknowledgements

This research was supported by the Deutsche Forschungsgemeinschaft (Ri191-19-1, Ri192-21-1, FOR 296/2-1). Pieces of this work are part of a Diploma thesis (S.H.). We would like to thank Joel K. Yisraeli and Froma Oberman for helpful advice with the UV crosslinking assay and Evita Mohr for assistance with the protein preparations.

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