Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.
RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.
Vasu
S
S
Section of Cell and Developmental Biology, Division of Biology 0347, University of California at San Diego, La Jolla, CA 92093, USA.
Shah
S
S
Orjalo
A
A
Park
M
M
Fischer
W H
WH
Forbes
D J
DJ
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D022022:Nuclear Pore / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014714:Vertebrates; D014981:Xenopus; D029867:Xenopus Proteins
D015778:Minor Histocompatibility Antigens; C512772:NUP107 protein, human; C440250:NUP133 protein, human; C079377:NUP153 protein, human; C440251:NUP160 protein, human; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C088025:Nup107 protein, rat; C513084:Nup153 protein, mouse; C513083:Nup153 protein, rat; C499080:Nup160 protein, Xenopus; D010446:Peptide Fragments; D012333:RNA, Messenger; D029867:Xenopus Proteins; C119239:nuclear pore complex protein 96; C093350:nuclear pore complex protein 98