The Interaction between Cap-binding Complex and RNA Export Factor Is Required for Intronless mRNA Export (2007)

RNA export factor (REF) is a component of the exon junction complex (EJC) that is deposited on mRNA in a splicing-dependent manner, and targets spliced mRNA for export. In this study, analysis of the RNA-binding protein complexes revealed that REF associates with β-globin mRNA at the region other than the EJC deposition site. Comparison between RNA polymerase II and T7 transcription and further analysis showed that the deposition of REF apart from the EJC is dependent on the 5′ cap structure, but not splicing. Excess amounts of m7GpppG cap analog reduced REF binding to intronless mRNA, and a co-immunoprecipitation experiment revealed that REF interacts with the cap-binding protein CBP20. The export of Cy3-labeled intronless β-globin mRNA from nuclei of HeLa cells was enhanced by co-injection of CBP20 and REF. Thus, REF recruited by CBP20 may play a stimulatory role to export the capped intronless mRNAs. RNA export factor (REF) is a component of the exon junction complex (EJC) that is deposited on mRNA in a splicing-dependent manner, and targets spliced mRNA for export. In this study, analysis of the RNA-binding protein complexes revealed that REF associates with β-globin mRNA at the region other than the EJC deposition site. Comparison between RNA polymerase II and T7 transcription and further analysis showed that the deposition of REF apart from the EJC is dependent on the 5′ cap structure, but not splicing. Excess amounts of m7GpppG cap analog reduced REF binding to intronless mRNA, and a co-immunoprecipitation experiment revealed that REF interacts with the cap-binding protein CBP20. The export of Cy3-labeled intronless β-globin mRNA from nuclei of HeLa cells was enhanced by co-injection of CBP20 and REF. Thus, REF recruited by CBP20 may play a stimulatory role to export the capped intronless mRNAs. In eukaryotic cells, pre-mRNAs are primarily transcribed by RNA polymerase II (RNAPII); this process is followed by complex RNA processing steps that include capping, splicing, and polyadenylation to produce mature mRNAs. Recent studies have shown that individual events occurring during eukaryotic gene expression are coupled together under more elaborate regulatory controls than previously imagined (1Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429Crossref PubMed Google Scholar, 2Howe K.J. Biochim. Biophys. Acta. 2002; 1577: 308-324Crossref PubMed Scopus (80) Google Scholar, 3Maniatis T. Reed R. Nature. 2002; 416: 499-506Crossref PubMed Scopus (928) Google Scholar, 4Neugebauer K.M. J. Cell Sci. 2002; 115: 3865-3871Crossref PubMed Scopus (240) Google Scholar, 5Kornblihtt A.R. Mata M. Fededa J.P. Munoz M.J. Nogues G. RNA (Cold Spring Harbor). 2004; 10: 1489-1498Google Scholar). Coupling stimulates the rate and specificity of enzymatic reactions by tethering mechanisms to each other and to their substrates. Following processing, mRNA is exported as a large mRNA-protein complex (mRNP) 2The abbreviations used are: mRNP, mRNA-protein complex; CBC, cap-binding complex; CBP20, cap-binding protein 20; EJC, exon-junction complex; AdMLP, adenovirus major late promoter; REF, RNA and export factor-binding proteins; RNAPII, RNA polymerase II; TREX, transcription/export (complex); nt, nucleotide(s); FITC, fluorescein isothiocyanate; RNP, ribonucleoprotein; WT, wild type. through the nuclear pore to the cytoplasm for subsequent translation. The transport of mRNA from the nucleus to the cytoplasm is linked to pre-mRNA splicing, especially in metazoans (6Luo M.L. Reed R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14937-14942Crossref PubMed Scopus (304) Google Scholar). Exon junction complexes (EJCs), which are deposited on mRNAs at specific sites relative to the exon junction as a consequence of splicing, form the basis of this connection (7Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar, 8Le Hir H. Gatfield D. Izaurralde E. Moore M.J. EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (612) Google Scholar). The EJC consists of four core proteins, eIF4A3 (9Chan C.C. Dostie J. Diem M.D. Feng W. Mann M. Rappsilber J. Dreyfuss G. RNA (Cold Spring Harbor). 2004; 10: 200-209Google Scholar, 10Palacios I.M. Gatfield D. St. Johnston D. Izaurralde E. Nature. 2004; 427: 753-757Crossref PubMed Scopus (300) Google Scholar, 11Shibuya T. Tange T.O. Sonenberg N. Moore M.J. Nat. Struct. Mol. Biol. 2004; 11: 346-351Crossref PubMed Scopus (218) Google Scholar), Y14 (7Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar), Magoh (12Le Hir H. Gatfield D. Braun I.C. Forler D. Izaurralde E. EMBO Rep. 2001; 2: 1119-1124Crossref PubMed Scopus (144) Google Scholar, 13Kataoka N. Diem M.D. Kim V.N. Yong J. Dreyfuss G. EMBO J. 2001; 20: 6424-6433Crossref PubMed Scopus (173) Google Scholar), and MLN51 (14Degot S. Le Hir H. Alpy F. Kedinger V. Stoll I. Wendling C. Seraphin B. Rio M.C. Tomasetto C. J. Biol. Chem. 2004; 279: 33702-33715Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), plus other auxiliary proteins including REF (7Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar), UAP56 (15Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (308) Google Scholar), RNPS1 (7Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar), SRm160 (7Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar), Pinin (16Li C. Lin R.I. Lai M.C. Ouyang P. Tarn W.Y. Mol. Cell. Biol. 2003; 23: 7363-7376Crossref PubMed Scopus (58) Google Scholar, 17Sakashita E. Tatsumi S. Werner D. Endo H. Mayeda A. Mol. Cell. Biol. 2004; 24: 1174-1187Crossref PubMed Scopus (82) Google Scholar), Acinus L (18Tange T.O. Shibuya T. Jurica M.S. Moore M.J. RNA (Cold Spring Harbor). 2005; 11: 1869-1883Google Scholar), SAP18 (18Tange T.O. Shibuya T. Jurica M.S. Moore M.J. RNA (Cold Spring Harbor). 2005; 11: 1869-1883Google Scholar), and hUpf3 (8Le Hir H. Gatfield D. Izaurralde E. Moore M.J. EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (612) Google Scholar, 19Lykke-Andersen J. Shu M.D. Steitz J.A. Cell. 2000; 103: 1121-1131Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 20Kim V.N. Kataoka N. Dreyfuss G. Science. 2001; 293: 1832-1836Crossref PubMed Scopus (246) Google Scholar). The recruitment of REF during mRNA biogenesis is thought to be responsible for the increased export of spliced mRNA (21Zhou Z. Luo M.J. Strasser K. Katahira J. Hurt E. Reed R. Nature. 2000; 407: 401-405Crossref PubMed Scopus (402) Google Scholar). A DEAD box RNA helicase, UAP56, is required for the recruitment of REF to mRNA (15Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (308) Google Scholar, 22Strasser K. Hurt E. Nature. 2001; 413: 648-652Crossref PubMed Scopus (223) Google Scholar). Subsequently, UAP56 is displaced from REF by the mRNA export factor TAP (23Stutz F. Bachi A. Doerks T. Braun I.C. Seraphin B. Wilm M. Bork P. Izaurralde E. RNA (Cold Spring Harbor). 2000; 6: 638-650Google Scholar). TAP forms a heterodimer with p15 that then directly interacts with the nuclear pore to facilitate mRNP transport into the cytoplasm (24Katahira J. Sträßer K. Podtelejnikov A. Mann M. Jung J.U. Hurt E. EMBO J. 1999; 18: 2593-2609Crossref PubMed Scopus (344) Google Scholar). Although the above model explains the apparent link between splicing and RNA export in metazoans, the question of how intronless mRNAs, which lack EJC deposition, are exported to the cytoplasm naturally arises. Some intronless transcripts (e.g. histone H2A) have been reported to contain specific sequences that recruit export factors independently of splicing (25Huang Y. Wimler K.M. Carmichael G.G. EMBO J. 1999; 18: 1642-1652Crossref PubMed Scopus (95) Google Scholar). SRp20 and 9G8, which belong to members of the evolutionarily conserved SR (serine/arginine-rich) protein family, specifically bind to a sequence in intronless mRNA and greatly facilitate the export of mRNA by recruiting TAP (26Huang Y. Steitz J.A. Mol. Cell. 2001; 7: 899-905Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). However, the intronless mRNAs coding Ftz, dihydrofolate reductase, and β-globin, which lack such cis-acting sequences, can be effectively exported regardless of whether splicing has occurred (27Rodrigues J.P. Rode M. Gatfield D. Blencowe B.J. Carmo-Fonseca M. Izaurralde E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1030-1035Crossref PubMed Scopus (218) Google Scholar, 28Ohno M. Segref A. Kuersten S. Mattaj I.W. Mol. Cell. 2002; 9: 659-671Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 29Masuyama K. Tanigushi I. Kataoka N. Ohno M. Genes Dev. 2004; 18: 2074-2085Crossref PubMed Scopus (66) Google Scholar). The injection experiments of α-REF antibody into the nuclei of Xenopus oocytes indicated that REF stimulates directly the export of these intronless mRNAs (27Rodrigues J.P. Rode M. Gatfield D. Blencowe B.J. Carmo-Fonseca M. Izaurralde E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1030-1035Crossref PubMed Scopus (218) Google Scholar). Mass spectrometry and Western blotting of purified spliceosomes revealed that REF is a component of H complex (30Reichert V.L. Le Hir H. Jurica M.S. Moore M.J. Genes Dev. 2002; 16: 2778-2791Crossref PubMed Scopus (117) Google Scholar), suggesting that REF can associate with mRNAs in a splicing-independent manner. In situ analysis of green fluorescent protein-tagged REF showed its accumulation at sites of transcription (31Custodio N. Carvalho C. Condado I. Antoniou M. Blencowe B.J. Carmo-Fonseca M. RNA (Cold Spring Harbor). 2004; 10: 622-633Google Scholar), suggesting that REF binds to mRNA co-transcriptionally. To clarify the recruiting mechanism of RNPs on mRNAs, we primarily developed a coupled in vitro transcription-splicing system. This in vitro system led us to discover a novel mechanism through which REF can associate with mRNA in a manner that is independent of splicing, instead of via the cap structure that is created during RNAPII transcription. The cap structure at the 5′ end of mRNA is associated with the nuclear cap-binding complex (CBC), consisting of CBP20 and CBP80 (32Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar). The microinjection of mRNAs into the living cell nuclei indicated that interaction between CBP20 and REF is required for the promotion of intronless mRNA export. Antibodies and Plasmids—The α-heterogeneous nuclear ribonucleoprotein A1 (4B10; Abcam), α-Y14 (4C4; Abcam), α-Aly/REF (11G5; Abcam), α-FLAG (M2; Sigma), and α-m3G/m7G cap (H20; Synaptic Systems) antibodies were purchased. The α-UAP56 and α-CBP20 antibodies were kindly provided by M. Green and E. Izaurralde, respectively. The β-globin Δ6 and pAd-SX (Eco) DNA templates were kindly provided by A. Krainer and K. Mizumoto, respectively. The DNA for the intronless mRNA was the In and in vitro splicing reactions were as by Krainer A.R. T. B. Green Cell. Full Text PDF PubMed Scopus Google Scholar). the coupled the and to and a of the β-globin was in a and of and HeLa nuclear The was from the region of the to of exon The was at for the indicated The were purified by protein and The purified were and a of DNA by the on the DNA at a a sequence in β-globin exon the were with of as by and Z. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The were then with and in a and at for the were with and in and RNA were to in vitro reactions and at for to RNA by H as previously by T. Shu M.D. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). was used to the mRNA from The RNA from the were with the indicated and on of was with HeLa nuclear in at for and then with The proteins to the were then to followed by Western was α-FLAG as in the to cell from cells were a The proteins were with A at for to α-FLAG antibody was used as the microinjection was as previously K. Shibuya T. Y. H. M. M. T. Y. T. Genes Cell. 11: PubMed Scopus Google Scholar). β-globin and 5′ m7GpppG cap and sequence was transcribed by T7 RNA The RNA was with the The of coupled with mRNA was at of The RNA purified protein and were into HeLa cell and at the of injection and injection The cells were at under for and under a with The of and were by with and respectively. REF to mRNA of the EJC in mRNP during mRNA biogenesis in the we developed a coupled in vitro system for transcription-splicing and DNA were as transcription templates to complexes transcripts for the EJC proteins UAP56, and are at sites of transcription (31Custodio N. Carvalho C. Condado I. Antoniou M. Blencowe B.J. Carmo-Fonseca M. RNA (Cold Spring Harbor). 2004; 10: 622-633Google Scholar), we in vitro transcribed and spliced β-globin mRNA antibodies the EJC and whether RNAPII transcription the EJC deposition The α-REF antibody has been reported to spliced mRNAs (21Zhou Z. Luo M.J. Strasser K. Katahira J. Hurt E. Reed R. Nature. 2000; 407: 401-405Crossref PubMed Scopus (402) Google Scholar). experiments revealed that the α-REF antibody the spliced form of the RNAPII transcripts but not the spliced form of the T7 transcripts and the other the α-heterogeneous nuclear ribonucleoprotein A1 antibody the and spliced as as the transcripts of RNAPII and T7 and with a that the H complex nuclear ribonucleoprotein A1 was on the T7 transcripts R. K. B. M. M. Reed R. Genes Dev. 20: PubMed Scopus Google Scholar). the that RNAPII transcription stimulates the recruitment of REF to the spliced To whether REF is recruited as EJC we the EJC deposition on spliced mRNA from T7 and RNAPII The EJC is specifically of the junction of spliced mRNAs (7Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (715) Google Scholar). a to was of during which the in vitro transcription-splicing was by the T7 the spliced β-globin mRNA to be to suggesting that the EJC on and spliced mRNAs not the between the EJC and the region of the spliced mRNA was by the of RNA from β-globin gene in the coupled in vitro transcription-splicing EJC at of the exon (8Le Hir H. Gatfield D. Izaurralde E. Moore M.J. EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (612) Google Scholar). Thus, we that the EJC be to on the and but not on the The co-immunoprecipitation of RNA from the β-globin templates in vitro revealed that each REF RNA more than RNA and in EJC were to associate at the EJC binding site. were more than RNA the REF was and in the amounts of and were with more than that REF and UAP56 have binding further of to as the relative to the exon junction of REF with binds to mRNA independently of splicing (27Rodrigues J.P. Rode M. Gatfield D. Blencowe B.J. Carmo-Fonseca M. Izaurralde E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1030-1035Crossref PubMed Scopus (218) Google Scholar). To whether the binding of REF to the is a β-globin was used in the coupled in vitro transcription-splicing and RNA by H were by α-REF H revealed that REF binds to intronless mRNA of the EJC deposition the of independent mechanisms of REF a splicing-dependent mechanism that of the exon junction to and that the EJC, and a splicing-independent mechanism at the site. of REF with is a RNA processing that is coupled to RNAPII transcription. that α-REF antibody RNAPII than T7 transcripts and and REF associated with spliced and intronless transcripts us to the of the cap structure in system. RNAPII transcripts were with the cap antibody more than the T7 transcripts This was with that is coupled to RNAPII transcription K. N. B. S. Mol. Cell. 2002; 10: Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. Mol. Cell. 2002; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). to the mechanism by which the cap structure is linked to REF we a in vitro splicing system and β-globin antibody REF revealed that REF associates with spliced and intronless mRNAs under in vitro splicing transcription is not coupled (27Rodrigues J.P. Rode M. Gatfield D. Blencowe B.J. Carmo-Fonseca M. Izaurralde E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1030-1035Crossref PubMed Scopus (218) Google Scholar). To that recruitment of REF is in the in vitro splicing H was to the site. This analysis revealed that REF to the of the spliced and the intronless mRNA in the of with RNAPII transcription not that the cap structure, than RNAPII transcription a role in REF recruitment at the site. The cap structure is by a nuclear that consists of CBP20 and CBP80 (32Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar). To the interaction between the cap structure and REF, we a with cap analog The from HeLa nuclear CBP20 and REF but not contain of the EJC and the protein not This interaction at in the of suggesting that RNA the proteins and that REF interacts with the cap structure and with to the of from cell the other and were with that REF and the associate with the cap structure not REF to we a co-immunoprecipitation analysis to the interaction between and REF. was into cells and then α-FLAG antibody from cell A Western of the revealed that CBP20 but not Y14 not was with a was for the co-immunoprecipitation CBP20, REF, Y14 was in the not these co-immunoprecipitation experiments showed that REF interacts with the in To whether the cap structure is for the intronless RNA binding of REF, we a experiment a cap analog shown in amounts of the cap analog the co-immunoprecipitation of the capped intronless RNA with REF, suggesting that the cap structure is required for interaction between REF and intronless whether the cap structure was required for of REF with spliced the in vitro splicing was by the of the cap analog as reported previously Cell. Full Text PDF PubMed Scopus Google Scholar), the spliced β-globin were with α-REF antibody The relative of the co-immunoprecipitation of spliced RNA in the of the cap analog was The in can be by the of REF recruitment from the of the cap but the splicing-dependent recruitment of REF into the EJC was not the of the cap analog RNA not The of UAP56 with intronless RNA was by the cap analog but the with spliced mRNA at a UAP56 not bind to the cap analog and the binding of UAP56 to intronless RNA was by the cap analog that UAP56 recruitment to intronless RNA may the binding of REF to The mRNA the region of REF, we expression of which lack of conserved as and (23Stutz F. Bachi A. Doerks T. Braun I.C. Seraphin B. Wilm M. Bork P. Izaurralde E. RNA (Cold Spring Harbor). 2000; 6: 638-650Google Scholar). The co-immunoprecipitation experiment from the cell with the expression showed sequences to be RNA-binding that are for the interaction with CBP20 and sequences are not in the with UAP56, and TAP not that sequences are for CBP20, but not other interaction proteins to the role of the interaction in mRNA we purified REF and a REF region and used for microinjection into HeLa cell nuclei with Cy3-labeled intronless β-globin mRNA, CBP20, and as injection The and amounts of CBP20, and were on the Cy3-labeled intronless β-globin mRNA with CBP20 and was to cytoplasm in more than of cells the Cy3-labeled RNA with CBP20 and was in nuclei in more than of cells during the that the interaction between CBP20 and REF is required for the promotion of intronless mRNA export from the RNAPII transcripts are with RNA-binding the that mRNP during transcription may be linked to the of mRNA processing has In this study, we that RNA nuclear export factor REF was recruited to RNAPII transcripts more than to T7 analysis this to the of REF with capped mRNAs through are with the that REF can with pre-mRNA to other EJC UAP56, and are in spliceosomes (30Reichert V.L. Le Hir H. Jurica M.S. Moore M.J. Genes Dev. 2002; 16: 2778-2791Crossref PubMed Scopus (117) Google Scholar). this may that REF proteins the export of spliced and intronless mRNAs from the nucleus (27Rodrigues J.P. Rode M. Gatfield D. Blencowe B.J. Carmo-Fonseca M. Izaurralde E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1030-1035Crossref PubMed Scopus (218) Google Scholar). REF was at in the transcripts a cap The α-REF antibody may be to REF in the complex under this The 5′ cap structure, a for CBC, stimulates pre-mRNA splicing M. Segref A. Kuersten S. Mattaj I.W. Mol. Cell. 2002; 9: 659-671Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 29Masuyama K. Tanigushi I. Kataoka N. Ohno M. Genes Dev. 2004; 18: 2074-2085Crossref PubMed Scopus (66) Google and polyadenylation P. Izaurralde E. Mattaj I.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google in the A in has shown that the is for the of the J. K.M. K. K.M. Mol. Cell. 2005; 19: Full Text Full Text PDF PubMed Scopus Google and that reduced the of 5′ sites but not that of sites Izaurralde E. A. McGuigan C. Mattaj I.W. Genes Dev. 10: PubMed Scopus Google Scholar). that the forms a complex on interaction between REF and the and the of a cap analog on the interaction between REF and capped RNA that the complex at and REF. The is for nuclear RNA export in metazoans E. Lewis J. C. A. McGuigan C. Mattaj I.W. Nature. PubMed Scopus Google Scholar). nuclear RNA export that is the but not the mRNA export TAP M. Ohno M. M. Mattaj I.W. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). for RNA as that the RNA complex to the complex M. Segref A. Bachi A. Wilm M. Mattaj I.W. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the export of nuclear RNA can be to a by the of a mRNA sequence into nuclear RNA M. Segref A. Kuersten S. Mattaj I.W. Mol. Cell. 2002; 9: 659-671Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the complex may as its mRNA export on the of the A model for the export of intronless mRNAs was on the export of intronless mRNAs M.D. Izaurralde E. S. Mattaj I.W. Kuersten S. EMBO J. 2001; 20: PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google Scholar). the protein that is in the export of mRNAs. The interaction of with REF was in a system and in cells M.D. Izaurralde E. S. Mattaj I.W. Kuersten S. EMBO J. 2001; 20: PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google Scholar). The injection of into Xenopus oocytes the export of intronless mRNAs, a that not with REF was during RNA export J. 2002; PubMed Scopus Google Scholar), that the recruitment of REF is for intronless mRNA export. The of REF in the export of intronless mRNAs was by experiments the injection of α-REF antibody into the nuclei of Xenopus oocytes M. Segref A. Kuersten S. Mattaj I.W. Mol. Cell. 2002; 9: 659-671Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). be by that REF was recruited by CBP20. the interaction was for the promotion of the intronless mRNA to microinjection experiments with and REF In cells, RNA experiments showed that are for mRNA export D. Izaurralde E. J. Cell Biol. 2002; PubMed Scopus Google Scholar), suggesting that mRNAs not REF for their export. the EJC is not on intronless mRNA, REF recruitment may play a role for the export of intronless mRNAs. In the complex consisting of and S. M. F. A. Mol. Cell. Biol. 2001; PubMed Scopus Google interacts and with of mRNA export S. R. A. EMBO J. 2002; PubMed Scopus Google Scholar, D. P. F. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). and are associated with the and is recruited to with and the transcription/export complex K. S. P. J. M. S. A. K. Reed R. Hurt E. Nature. 2002; PubMed Scopus Google Scholar, S. M. EMBO J. 2004; 23: PubMed Scopus Google Scholar). In the of gene expression in cells of UAP56, and REF showed that play in mRNA suggesting these proteins not as of a protein complex D. Izaurralde E. J. Cell Biol. 2002; PubMed Scopus Google Scholar, A. Izaurralde E. EMBO J. 2003; PubMed Scopus Google Scholar, J. A. K. T. Rode M. Wilm M. Izaurralde E. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). In and REF in HeLa nuclear S. R. H. Hurt E. N. Reed R. Genes Dev. 2005; 19: PubMed Scopus Google Scholar). S. R. H. Hurt E. N. Reed R. Genes Dev. 2005; 19: PubMed Scopus Google to the complex REF and UAP56 as the However, pre-mRNA splicing and the 5′ cap structure are required for the recruitment of complex to mRNA, and this recruitment not on RNAPII H. K. J.L. A. Reed R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, in mechanisms of REF recruitment to mRNA may splicing-dependent recruitment the EJC, and splicing-dependent recruitment the complex on spliced mRNA, and recruitment to intronless Although further studies are may play in mRNA export in are to Green and for α-UAP56 antibody and expression and Izaurralde for the α-CBP20 and for and Kataoka for for the mRNA microinjection and members of the for with