Interactive reportTwo trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs1
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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Published on the World Wide Web on 3 July 2000.