RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains.
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.
Pritchard
C E
CE
Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Fornerod
M
M
Kasper
L H
LH
van Deursen
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003609:Dactinomycin / Q000494:pharmacology; D005260:Female; D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism* / Q000502:physiology; D034521:Nuclear Matrix-Associated Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D009865:Oocytes / Q000502:physiology; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014982:Xenopus laevis
D003432:Cross-Linking Reagents; D005656:Fungal Proteins; C490524:GLE2 protein, S cerevisiae; D008565:Membrane Proteins; D034521:Nuclear Matrix-Associated Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C490522:RAE1 protein, human; D012333:RNA, Messenger; C490521:Rae1 protein, mouse; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C093350:nuclear pore complex protein 98; C092953:rae1 protein, S pombe; D003609:Dactinomycin; D012319:RNA Polymerase II