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

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.

[1]  A. Podtelejnikov,et al.  The Mex67p‐mediated nuclear mRNA export pathway is conserved from yeast to human , 1999, The EMBO journal.

[2]  T. Rana,et al.  Tat stimulates cotranscriptional capping of HIV mRNA. , 2002, Molecular cell.

[3]  E. Izaurralde,et al.  Genome‐wide analysis of nuclear mRNA export pathways in Drosophila , 2003, The EMBO journal.

[4]  R. Luna,et al.  The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability , 2002, The EMBO journal.

[5]  Angela Bachi,et al.  PHAX, a Mediator of U snRNA Nuclear Export Whose Activity Is Regulated by Phosphorylation , 2000, Cell.

[6]  S. Lacadie,et al.  Biochemical analysis of TREX complex recruitment to intronless and intron‐containing yeast genes , 2004, The EMBO journal.

[7]  H. Le Hir,et al.  5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. , 2002, Genes & development.

[8]  N. Kataoka,et al.  RNA length defines RNA export pathway. , 2004, Genes & development.

[9]  S. Kuersten,et al.  Identity elements used in export of mRNAs. , 2002, Molecular cell.

[10]  E. Sakashita,et al.  Human RNPS1 and Its Associated Factors: a Versatile Alternative Pre-mRNA Splicing Regulator In Vivo , 2004, Molecular and Cellular Biology.

[11]  Kevin Struhl,et al.  TREX is a conserved complex coupling transcription with messenger RNA export , 2002, Nature.

[12]  D. Gatfield,et al.  An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay , 2004, Nature.

[13]  Joe D. Lewis,et al.  A nuclear cap binding protein complex involved in pre-mRNA splicing , 1994, Cell.

[14]  Seiji Masuda,et al.  Recruitment of the human TREX complex to mRNA during splicing. , 2005, Genes & development.

[15]  M. Lai,et al.  Nuclear Pnn/DRS Protein Binds to Spliced mRNPs and Participates in mRNA Processing and Export via Interaction with RNPS1 , 2003, Molecular and Cellular Biology.

[16]  C. McGuigan,et al.  A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site. , 1996, Genes & development.

[17]  M. Moore,et al.  eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay , 2004, Nature Structural &Molecular Biology.

[18]  Ed Hurt,et al.  Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p , 2001, Nature.

[19]  J. Manley,et al.  RNA polymerase II and the integration of nuclear events. , 2000, Genes & development.

[20]  J. Steitz,et al.  Human Upf Proteins Target an mRNA for Nonsense-Mediated Decay When Bound Downstream of a Termination Codon , 2000, Cell.

[21]  Daniel Zenklusen,et al.  Stable mRNP Formation and Export Require Cotranscriptional Recruitment of the mRNA Export Factors Yra1p and Sub2p by Hpr1p , 2002, Molecular and Cellular Biology.

[22]  Minoru Yoshida,et al.  CRM1 Is an Export Receptor for Leucine-Rich Nuclear Export Signals , 1997, Cell.

[23]  E. Hurt,et al.  The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans , 2000, Nature.

[24]  H. Le Hir,et al.  The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon–exon junctions , 2000, The EMBO journal.

[25]  M. Mann,et al.  Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly , 2001, Nature.

[26]  S. Kuersten,et al.  Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway , 2001, The EMBO journal.

[27]  K. Neugebauer,et al.  On the importance of being co-transcriptional , 2002, Journal of Cell Science.

[28]  P. Fortes,et al.  Participation of the nuclear cap binding complex in pre-mRNA 3' processing. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Gatfield,et al.  The protein Mago provides a link between splicing and mRNA localization , 2001, EMBO Reports.

[30]  J. Steitz,et al.  Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. , 2001, Molecular cell.

[31]  R. Reed,et al.  Human mRNA Export Machinery Recruited to the 5′ End of mRNA , 2006, Cell.

[32]  T. Maniatis,et al.  An extensive network of coupling among gene expression machines , 2002, Nature.

[33]  R. Reed,et al.  Splicing is required for rapid and efficient mRNA export in metazoans. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  R. Reed,et al.  Functional coupling of RNAP II transcription to spliceosome assembly. , 2006, Genes & development.

[35]  Y. Ohshima,et al.  Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: nuclear mRNA export is coupled to ongoing gene transcription , 2006, Genes to cells : devoted to molecular & cellular mechanisms.

[36]  Z. Wang,et al.  DNA damage-dependent transcriptional arrest and termination of RNA polymerase II elongation complexes in DNA template containing HIV-1 promoter. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Marie-Christine Rio,et al.  Association of the Breast Cancer Protein MLN51 with the Exon Junction Complex via Its Speckle Localizer and RNA Binding Module* , 2004, Journal of Biological Chemistry.

[38]  G. Dreyfuss,et al.  Role of the Nonsense-Mediated Decay Factor hUpf3 in the Splicing-Dependent Exon-Exon Junction Complex , 2001, Science.

[39]  K. J. Howe.  RNA polymerase II conducts a symphony of pre-mRNA processing activities. , 2002, Biochimica et biophysica acta.

[40]  D. Gatfield,et al.  REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export , 2002, The Journal of cell biology.

[41]  Michael R. Green,et al.  Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro , 1984, Cell.

[42]  J. Yong,et al.  Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing‐dependent exon–exon junction complex , 2001, EMBO Journal.

[43]  T. Köcher,et al.  Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster , 2004, Nature Structural &Molecular Biology.

[44]  P. Sharp,et al.  Recognition of cap structure in splicing in vitro of mRNA precursors , 1984, Cell.

[45]  D. Price,et al.  Functional coupling of capping and transcription of mRNA. , 2002, Molecular cell.

[46]  K. Neugebauer,et al.  Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex. , 2005, Molecular cell.

[47]  G. Carmichael,et al.  Intronless mRNA transport elements may affect multiple steps of pre‐mRNA processing , 1999, The EMBO journal.

[48]  S. Chávez,et al.  Hpr1 Is Preferentially Required for Transcription of Either Long or G+C-Rich DNA Sequences in Saccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

[49]  R. Sandri-Goldin,et al.  ICP27 Interacts with the RNA Export Factor Aly/REF To Direct Herpes Simplex Virus Type 1 Intronless mRNAs to the TAP Export Pathway , 2002, Journal of Virology.

[50]  J. Steitz,et al.  Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Joe D. Lewis,et al.  A cap-binding protein complex mediating U snRNA export , 1995, Nature.

[52]  H. Le Hir,et al.  The exon–exon junction complex provides a binding platform for factors involved in mRNA export and nonsense‐mediated mRNA decay , 2001, The EMBO journal.