Abstract
Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Binding Sites
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Cell Line
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Cricetinae
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Cross-Linking Reagents
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Dactinomycin / pharmacology
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Female
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Fungal Proteins / metabolism*
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Humans
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Mice
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Molecular Sequence Data
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Nuclear Envelope / metabolism*
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Nuclear Envelope / physiology
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Nuclear Matrix-Associated Proteins*
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Nuclear Pore Complex Proteins*
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Nucleocytoplasmic Transport Proteins*
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Oocytes / physiology
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RNA Polymerase II / metabolism
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RNA, Messenger / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Repetitive Sequences, Amino Acid
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins*
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Schizosaccharomyces pombe Proteins*
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Sequence Alignment
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Sequence Homology, Amino Acid
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Transfection
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Xenopus laevis
Substances
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Cross-Linking Reagents
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Fungal Proteins
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GLE2 protein, S cerevisiae
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Membrane Proteins
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Nuclear Matrix-Associated Proteins
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Nuclear Pore Complex Proteins
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Nuclear Proteins
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Nucleocytoplasmic Transport Proteins
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RAE1 protein, human
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RNA, Messenger
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Rae1 protein, mouse
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces pombe Proteins
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nuclear pore complex protein 98
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rae1 protein, S pombe
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Dactinomycin
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RNA Polymerase II