U2AF Participates in the Binding of TAP (NXF1) to mRNA

Abstract

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates. TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates. amino acid(s) cytoplasm nucleus nucleoplasm messenger ribonucleoprotein heterogeneous nuclear ribonucleoprotein small nuclear ribonucleoprotein 4-morpholinepropanesulfonic acid green fluorescent protein hemagglutinin glutathione S-transferase chloramphenicol acetyltransferase glyceraldehyde phosphate dehydrogenase luciferase constitutive transport element TAP (also called NXF1)/Mex67p proteins define a conserved mRNA export pathway in eukaryotes (1Bachi A. Braun I.C. Rodrigues J.P. Pante N. Ribbeck K. von Kobbe C. Kutay U. Wilm M. Gorlich D. Carmo-Fonseca M. Izaurralde E. RNA. 2000; 6: 136-158Crossref PubMed Scopus (275) Google Scholar, 2Braun I.C. Rohrbach E. Schmitt C. Izaurralde E. EMBO J. 1999; 18: 1953-1965Crossref PubMed Scopus (160) Google Scholar, 3Gruter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 4Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (436) Google Scholar). The Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans Ce-NXF-1 are essential for mRNA export (4Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (436) Google Scholar, 5Tan W. Zolotukhin A.S. Bear J. Patenaude D.J. Felber B.K. RNA. 2000; 6: 1762-1772Crossref PubMed Scopus (96) Google Scholar), and the vertebrate TAP mediates the export of mRNA, but not the other classes of RNA (3Gruter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar,6Pasquinelli A.E. Ernst R.K. Lund E. Grimm C. Zapp M.L. Rekosh D. Hammarskjold M.L. Dahlberg J.E. EMBO J. 1997; 16: 7500-7510Crossref PubMed Scopus (198) Google Scholar, 7Saavedra C. Felber B. Izaurralde E. Curr. Biol. 1997; 7: 619-628Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The Schizosaccharomyces pombe spMex67p is nonessential, but its mRNA export function is required in the absence of the essential Rae1p (8Brown J.A. Bharathi A. Ghosh A. Whalen W. Fitzgerald E. Dhar R. J. Biol. Chem. 1995; 270: 7411-7419Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Yoon J.H. Love D.C. Guhathakurta A. Hanover J.A. Dhar R. Mol. Cell. Biol. 2000; 20: 8767-8782Crossref PubMed Scopus (72) Google Scholar). TAP belongs to the NXF family of proteins that share the ability to bind both RNA and the components of the nuclear pore complex (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar). The proposed substrate binding domain of TAP (aa1 1–372) comprises an RNA binding domain and leucine-rich repeat domain and exhibits general RNA binding and high affinity binding to the viral CTE RNA (2Braun I.C. Rohrbach E. Schmitt C. Izaurralde E. EMBO J. 1999; 18: 1953-1965Crossref PubMed Scopus (160) Google Scholar, 3Gruter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 11Liker E. Fernandez E. Izaurralde E. Conti E. EMBO J. 2000; 19: 5587-5598Crossref PubMed Scopus (113) Google Scholar). This region is also required for the direct binding to REF (also called Aly) proteins (1Bachi A. Braun I.C. Rodrigues J.P. Pante N. Ribbeck K. von Kobbe C. Kutay U. Wilm M. Gorlich D. Carmo-Fonseca M. Izaurralde E. RNA. 2000; 6: 136-158Crossref PubMed Scopus (275) Google Scholar, 12Stutz F. Bachi A. Doerks T. Braun I.C. Seraphin B. Wilm M. Bork P. Izaurralde E. RNA. 2000; 6: 638-650Crossref PubMed Scopus (309) Google Scholar), which are the metazoan homologues of yeast mRNA export factor Yra1p (13Strasser K. Hurt E. EMBO J. 2000; 19: 410-420Crossref PubMed Google Scholar). At all metabolic steps, mRNA is part of ribonucleoprotein complexes (mRNP). The primary transcripts associate with a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) (reviewed in Ref. 14Nakielny Cell. 1999; Full Text Full Text PDF PubMed Scopus Google and with the splicing J. Cell. Full Text PDF PubMed Scopus Google Scholar, P. M. Cell. Full Text PDF PubMed Scopus Google Scholar), leading to nuclear the mRNP by the of mRNP R. U. A. 1999; PubMed Scopus Google Scholar, K. J. Hurt E. R. 2000; PubMed Scopus Google and by proteins Izaurralde E. EMBO J. 2000; 19: PubMed Scopus Google Scholar). This protein complex is of the and as as REF and the protein Izaurralde E. EMBO J. 2000; 19: PubMed Scopus Google Scholar, 2000; Google Scholar). REF proposed to a link to export, by TAP K. J. Hurt E. R. 2000; PubMed Scopus Google Scholar, J.P. M. D. Carmo-Fonseca M. Izaurralde E. U. A. PubMed Scopus Google Scholar, D. P. F. Mol. Cell. Biol. PubMed Scopus Google Scholar). in human TAP is added to the mRNP complexes N. J. N. F. F. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). TAP is not added to the complexes by vitro K. J. Hurt E. R. 2000; PubMed Scopus Google Scholar, Izaurralde E. EMBO J. 2000; 19: PubMed Scopus Google in (2Braun I.C. Rohrbach E. Schmitt C. Izaurralde E. EMBO J. 1999; 18: 1953-1965Crossref PubMed Scopus (160) Google Scholar). this we the of TAP with the mRNP in human cells. We that TAP is to the spliced a region that directly to U2AF35, the small subunit of the splicing factor U2AF. We further found that U2AF TAP to mRNP and is to the nuclear export of mRNA in in human and J. W. Zolotukhin A.S. Tabernero C. Felber B.K. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, A.S. A. Felber B.K. J. PubMed Google Scholar), export (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, D. U. A. PubMed Scopus Google Scholar), the chloramphenicol acetyltransferase luciferase and J. W. Zolotukhin A.S. Tabernero C. Felber B.K. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, A.S. Felber B.K. J. 1999; PubMed Google as to TAP (3Gruter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar), U2AF35 and U2AF65 P. T. PubMed Scopus Google Scholar), the PubMed Scopus Google Scholar), and to M. J. Biol. 1999; PubMed Scopus Google by E. T. and J. The and to the the from PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, and N. J. Biol. 1997; PubMed Scopus Google cells with Felber B.K. J.E. Mol. Cell. Biol. PubMed Scopus Google with The in the by and a and the in the of and the nuclear and in the of to all for and the RNA the and with a The for human glyceraldehyde phosphate dehydrogenase and of the and a The for the spliced and and a mRNP from cells to in and with of for with for to binding to the the in the the with by in U2AF expression the K. P. 1998; PubMed Scopus Google that a two-hybrid as W. Zolotukhin A.S. Bear J. Patenaude D.J. Felber B.K. RNA. 2000; 6: 1762-1772Crossref PubMed Scopus (96) Google Scholar). elegans U2AF35 T. RNA. 1999; PubMed Scopus Google Scholar), the expression for and TAP proteins J. W. Zolotukhin A.S. Tabernero C. Felber B.K. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar), and the expression for glutathione S-transferase and P. T. PubMed Scopus Google The expression for REF proteins and the expression by E. both RNA and A.S. A. Felber B.K. J. PubMed Google and luciferase and the D. U. A. PubMed Scopus Google Scholar). The We the of TAP with mRNP in The by in and the nuclear with and the of associate with the nuclear the by the to Ref. PubMed Scopus Google The of spliced and transcripts in these by for that the and in spliced mRNA, in as PubMed Scopus Google Scholar). The also small of spliced transcripts that not We further the transcripts in human cells by and found a of spliced and in the also these data that the are not the of a directly the nuclear export of mRNA in human cells. cells with of and of in the absence in the of of as the complexes with of from the and as the The RNA from the and from of the and for and The of the and the spliced are to the The RNA from the and for The of the are to with of in the absence in the of of of and of as The transcripts as in A. of the and the spliced transcripts in the from the in A. addition to the and the the to and that TAP with in the and Using we that in PubMed Scopus Google Scholar), in with the that the general N. J. N. F. F. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). We that proteins as and from the as N. J. Biol. 1997; PubMed Scopus Google Scholar, M. R. Biol. PubMed Scopus Google Scholar), the of the of TAP to and the complexes with RNA TAP is a mRNP This to also and not to that of proteins PubMed Scopus Google Scholar). the human of Rae1p A. Ghosh A. Whalen J.H. R. M. Dhar R. 1997; PubMed Google Scholar, D. U. A. 1997; PubMed Scopus Google Scholar), not in that the is and is not a of mRNA export We that the binding of TAP splicing is by protein a for we the mRNP region of TAP. TAP and its expressed in human cells as proteins and for mRNP in nuclear in the TAP, the expressed with in a from TAP with hemagglutinin also for the not that this an in vitro we added to the to this protein the domain of TAP, it not the that in and not bind its the binding and of TAP and in the region that further to this We that TAP is to mRNP a region for proteins that with the mRNP we a yeast two-hybrid of a to the domain of as Using we found that of primary also with the human TAP, its human (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, J. W. Zolotukhin A.S. Tabernero C. Felber B.K. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar), and the C. elegans Ce-NXF-1 (4Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (436) Google Scholar, 5Tan W. Zolotukhin A.S. Bear J. Patenaude D.J. Felber B.K. RNA. 2000; 6: 1762-1772Crossref PubMed Scopus (96) Google Scholar). with the the human nuclear export the further indicating of the that these a U2AF35 protein the small subunit of the splicing factor U2AF. Using we found that the C. elegans T. RNA. 1999; PubMed Scopus Google also with the human TAP, and The of TAP and are that of Ce-NXF-1 not share this interaction is conserved between proteins across metazoan interaction between metazoan TAP and region that of TAP from of by of the region the with U2AF35 the with the by J. E. R. Cell. Full Text PDF PubMed Scopus Google Scholar), to the between and the and are as not The region that of TAP from of J. W. Zolotukhin A.S. Tabernero C. Felber B.K. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar), by of the region in a The the with U2AF35 the with the by J. E. R. Cell. Full Text PDF PubMed Scopus Google Scholar), to the between and the and are as not U2AF35 and U2AF65, the large subunit of a domain of a family of that the TAP proteins with we show that which with all TAP proteins the in but not the interaction the domain of not the not the domain not the primary TAP interaction and the interaction is for We the interaction between TAP and U2AF35 proteins in vitro TAP proteins in E. as and the U2AF35 protein expressed in The of proteins and the glutathione and in and to that are that the not bind U2AF35, the of the We found that of to U2AF35 the to the and and of the and the that a of U2AF35 data show that TAP a direct binding site for U2AF35, which is between and and coincides with the mRNP region We the ability of to bind RNA. RNA directly J. K. A. M. Hurt E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). We the general RNA binding of a of TAP to a RNA (4Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (436) Google in a RNA binding site found between and RNA not RNA and U2AF35 bind to TAP we the in the of CTE RNA (3Gruter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google human mRNA (2Braun I.C. Rohrbach E. Schmitt C. Izaurralde E. EMBO J. 1999; 18: 1953-1965Crossref PubMed Scopus (160) Google Scholar). We to that the RNA binding site in the of the RNA binding domain Ref. 11Liker E. Fernandez E. Izaurralde E. Conti E. EMBO J. 2000; 19: 5587-5598Crossref PubMed Scopus (113) Google Scholar). to but not for binding the we which binding to both RNA and U2AF35, as and the mRNP region of TAP comprises direct binding for U2AF35 and RNA. U2AF is to in splicing and not associate with the in vitro Biol. 2000; 7: PubMed Scopus Google and R. Curr. Biol. 2000; PubMed Scopus Google Scholar). TAP is added splicing it is to mRNP its U2AF35 binding region and the of U2AF this is that the U2AF with TAP and TAP's addition to the spliced in a that is to a of U2AF spliced mRNA, the for TAP. U2AF associate with spliced in we the complexes from of these complexes by for and by for and transcripts that but not the from the further that U2AF35 with the spliced as as the transcripts and but the in the complexes with the The U2AF35 also mRNA of the transcripts that with and U2AF35 that the of the RNA is in the of the complexes that found in the complexes in the and both of which are in spliced mRNA also the of complexes the of we found that the exogenously expressed U2AF accumulates with the spliced mRNP in the cytoplasm that it with in this U2AF bind to directly EMBO J. PubMed Scopus Google Scholar), the in from and in to the direct binding and to that U2AF further this we show that the of complexes with RNA human U2AF in the absence of the with to both U2AF35 not with that our U2AF. the U2AF65 not these data that a of U2AF with spliced the of complexes are a and mRNA also with U2AF. We as a of the Izaurralde E. EMBO J. 2000; 19: PubMed Scopus Google to its with the human of that in the nuclear as as in the PubMed Scopus Google not with its with the nuclear components R. J.A. 1998; PubMed Scopus Google Scholar, T. E. R. E. J.A. D. J. J. Biol. 2000; PubMed Scopus Google Scholar). not in mRNP and not found in complexes the complexes and to these with the in vitro splicing of this data J. N. EMBO J. 20: PubMed Scopus Google show that as REF are with mRNA the as are the of U2AF the of TAP with mRNP, we the TAP with the U2AF in human cells and mRNP in nuclear and and We found that U2AF65 in mRNP. The expression of U2AF65 to recruitment of the U2AF35 and to these complexes the and the not other we identified the and proteins which are of the protein complex B.K. R. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, A. P. K. B. J. Biol. 1999; PubMed Scopus Google Scholar). is that direct binding to it is to directly bind U2AF in vitro J. R. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). The not the the expression of U2AF65 not the splicing and nuclear of indicating that of general to the nuclear U2AF spliced mRNP. The expression of U2AF35 not to the of U2AF and in U2AF65 the of U2AF and U2AF35 by to RNA PubMed Scopus Google Scholar, M. Carmo-Fonseca M. U. A. PubMed Scopus Google Scholar), these that U2AF to mRNA directly U2AF65 and TAP direct binding to the U2AF35 The expression of U2AF65 also to the of but not of from the nuclear of TAP's to the not cells high of U2AF65, a of this protein with in an the of with by high U2AF65 that to the but not to the U2AF. We the of REF proteins TAP's with mRNP. we the and which and expressed in cells to to of the to TAP and mRNA F. Bachi A. Doerks T. Braun I.C. Seraphin B. Wilm M. Bork P. Izaurralde E. RNA. 2000; 6: 638-650Crossref PubMed Scopus (309) Google Scholar), not in mRNP in to the and expression the REF proteins to as to export K. J. Hurt E. R. 2000; PubMed Scopus Google J.P. M. D. Carmo-Fonseca M. Izaurralde E. U. A. PubMed Scopus Google Scholar), it is that this of is by the of data suggest that the recruitment of TAP is and is not a of that affinity to TAP and to RNA. This is with our finding that TAP the of the U2AF35 but not the REF 1–372) binding region required for the between TAP and mRNP these that TAP to the U2AF in We the of TAP in mRNP complexes nuclear TAP with mRNP in N. J. N. F. F. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google and this the in vitro and not its mRNA TAP (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, I.C. A. M. Conti E. Izaurralde E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google to the otherwise expression of which an D. U. A. PubMed Scopus Google Scholar). this TAP is a factor for the export of an mRNA that is otherwise retained in the nucleus, but not for exported (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, I.C. A. M. Conti E. Izaurralde E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google Scholar). the expression of TAP to a small of the of TAP's to further (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, I.C. A. M. Conti E. Izaurralde E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Rekosh D. Hammarskjold M.L. Mol. Cell. Biol. PubMed Scopus Google Scholar). the is a of with which to the TAP's this and of human cells with a of and of the The of TAP to a of but expression of the luciferase mRNA and of further the as (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, I.C. A. M. Conti E. Izaurralde E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Rekosh D. Hammarskjold M.L. Mol. Cell. Biol. PubMed Scopus Google Scholar). We the the that to the of TAP in mRNP. of and TAP with of U2AF65, we a of but not of the with that with TAP the expression of U2AF65 both TAP and of U2AF65 further We U2AF65 the of this is the TAP is as in the absence of further the of U2AF65 nuclear export, we in the absence in the of TAP, and and the transcripts it spliced as the absence of exogenously expressed nuclear the spliced mRNA exported as D. U. A. PubMed Scopus Google Scholar). expression of TAP to a of export, which further stimulated of expression of U2AF65 also in the of in the cytoplasm and and of TAP further the of U2AF65 to a of This is to the of human export Felber B.K. J. PubMed Google Scholar). proposed for this a of the but this not further The of U2AF65 for the of the luciferase mRNA the and not transcripts not U2AF65 not the protein from the exported transcripts as and not to TAP (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, I.C. A. M. Conti E. Izaurralde E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google Scholar). the of nuclear not by U2AF65 its to nuclear by the nuclear but to its this export direct the of the of nuclear we the complexes from the cells that the and the that with both in the nucleus and in the its with and the a of is to export, in the cytoplasm to the exported mRNA in the nucleus, its export not further the that in this the the exported as is that the transcripts as are exported and and U2AF65 are not in this that the of U2AF65 by its general with to the of the of factor M. PubMed Scopus Google Scholar), we the spliced transcripts U2AF65 not splicing not to the of to the and that is general with nuclear these the transcripts not further the of U2AF65 we the the and to nuclear to that of but the expression of U2AF65 not these the is to it also in the in the of U2AF65 its general these in the the as as the in the of U2AF65, to nuclear we the of this the of in the also to its nuclear in but these not further these data that U2AF65 the export of our in human U2AF65 the nuclear of by directly its nuclear the U2AF65 TAP to the general mRNP complexes and TAP with U2AF65 to the expression it is that the of U2AF65 is by TAP. we that U2AF TAP to mRNP complexes and directly the export of mRNA, which is otherwise retained in the nucleus. The mRNP of TAP not its high affinity CTE binding site the that TAP to with CTE and with the (2Braun I.C. Rohrbach E. Schmitt C. Izaurralde E. EMBO J. 1999; 18: 1953-1965Crossref PubMed Scopus (160) Google Scholar, 3Gruter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, 11Liker E. Fernandez E. Izaurralde E. Conti E. EMBO J. 2000; 19: 5587-5598Crossref PubMed Scopus (113) Google Scholar). We found that TAP is to the spliced mRNP a region that directly to U2AF35 subunit, and this interaction is conserved across metazoan This link of U2AF to nuclear export is further by our finding that both U2AF between the nucleus and the cytoplasm not an that by M. A. J. Carmo-Fonseca M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). both U2AF35 and U2AF65 to a of family with a proposed in nuclear export 1998; PubMed Scopus Google Scholar). our of the mRNP region of TAP not the of it in to TAP's which not by our the and not it to in of this a that REF is from nuclear in J. N. EMBO J. 20: PubMed Scopus Google Scholar). to REF a and not a link between TAP and At it is not REF and U2AF are part of the mechanism of TAP's the mRNP region of TAP affinity to it not the conserved RNA binding domain and of its E. Fernandez E. Izaurralde E. Conti E. EMBO J. 2000; 19: 5587-5598Crossref PubMed Scopus (113) Google Scholar). proteins conserved affinity to RNA and to RNA in TAP mRNA directly is that TAP to protein in mRNP and further the direct with RNA. Using human we show that splicing the of U2AF from TAP with spliced mRNP we the of U2AF with in both the human and in the complexes in not U2AF also found that the as the spliced mRNA and the the metabolic which the interaction is that TAP to U2AF in spliced to U2AF with TAP in and its addition to in a that is to to the addition of Izaurralde E. EMBO J. 2000; 19: PubMed Scopus Google Scholar, 2000; Google Scholar). the is to the export function of we nuclear with U2AF Using we found that U2AF with the and the of mRNA, as as with but not with and and the spliced U2AF the splicing of but the of the and the splicing as as these of the not not that U2AF is not for the nuclear export of mRNA in and the to further its export We found in the exogenously expressed U2AF65 in spliced leading to the recruitment of U2AF35 and TAP, which is with the direct binding of TAP to U2AF. also with U2AF these but the export of exported not further stimulated by U2AF65, which is in with the finding that TAP is not for the that are exported (10Herold A. Suyama M. Rodrigues J.P. Braun I.C. Kutay U. Carmo-Fonseca M. Bork P. Izaurralde E. Mol. Cell. Biol. 2000; 20: 8996-9008Crossref PubMed Scopus (183) Google Scholar, I.C. A. M. Conti E. Izaurralde E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google Scholar). the export of an otherwise is by U2AF65 with export, further a direct We found a between the and export by U2AF65 stimulated the export, but not the expression of TAP in the of U2AF65 not further export, but to an of expression and a TAP is a of the mRNP complexes and not it is that TAP a U2AF the export of mRNA, and with TAP to its it a for transcripts that are to nuclear this export with a mechanism by which TAP is added to spliced this TAP is to with U2AF and is as a of At of U2AF65, this splicing the of TAP as leading to export The of in vitro that the addition of TAP to spliced complexes in the of the We T. E. T. J. and N. for and D. for in and T. for The protein the by of