Adaptor Aly and co‐adaptor Thoc5 function in the Tap‐p15‐mediated nuclear export of HSP70 mRNA

In metazoans, nuclear export of bulk mRNA is mediated by Tap‐p15, a conserved heterodimeric export receptor that cooperates with adaptor RNA‐binding proteins. In this article, we show that Thoc5, a subunit of the mammalian TREX complex, binds to a distinct surface on the middle (Ntf2‐like) domain of Tap. Notably, adaptor protein Aly and Thoc5 can simultaneously bind to non‐overlapping binding sites on Tap‐p15. In vivo, Thoc5 was not required for bulk mRNA export. However, nuclear export of HSP70 mRNA depends on both Thoc5 and Aly. Consistent with a function as a specific export adaptor, Thoc5 exhibits in vitro RNA‐binding activity and is associated with HSP70 mRNPs in vivo as a component of the stable THO complex. Thus, through the combinatorial use of an adaptor (e.g., Aly) and co‐adapter (e.g., Thoc5), Tap‐p15 could function as an export receptor for different classes of mRNAs.

[1]  Ed Hurt,et al.  Exporting RNA from the nucleus to the cytoplasm , 2007, Nature Reviews Molecular Cell Biology.

[2]  L. J. Terry,et al.  Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex , 2007, The Journal of cell biology.

[3]  M. Künzler,et al.  Cse1p functions as the nuclear export receptor for importin alpha in yeast. , 1998, FEBS letters.

[4]  Robert Tjian,et al.  IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. , 2007, Genes & development.

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

[6]  Ed Hurt,et al.  Binding of the Mex67p/Mtr2p Heterodimer to Fxfg, Glfg, and Fg Repeat Nucleoporins Is Essential for Nuclear mRNA Export , 2000, The Journal of cell biology.

[7]  T. Miki,et al.  NXF2 is involved in cytoplasmic mRNA dynamics through interactions with motor proteins , 2007, Nucleic acids research.

[8]  Ed Hurt,et al.  A Conserved mRNA Export Machinery Coupled to pre-mRNA Splicing , 2002, Cell.

[9]  Narayanan Eswar,et al.  Simple fold composition and modular architecture of the nuclear pore complex , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Abel R. Alcázar-Román,et al.  Interaction between the shuttling mRNA export factor Gle1 and the nucleoporin hCG1: a conserved mechanism in the export of Hsp70 mRNA. , 2005, Molecular biology of the cell.

[11]  Mary K. Young,et al.  Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors , 2007, Nucleic acids research.

[12]  J. Cáceres,et al.  The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans. , 2003, RNA.

[13]  P. Bork,et al.  REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. , 2000, RNA.

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

[15]  M. Wilm,et al.  TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. , 1998, Molecular cell.

[16]  A. De Benedetti,et al.  Preferential translation of heat shock mRNAs in HeLa cells deficient in protein synthesis initiation factors eIF-4E and eIF-4 gamma. , 1992, Journal of Biological Chemistry.

[17]  D. Hultmark,et al.  Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader. , 1985, The EMBO journal.

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

[19]  D. Hultmark,et al.  Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22 , 1986, Cell.

[20]  R. Lührmann,et al.  Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores , 1997, The EMBO journal.

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

[22]  J. Steitz,et al.  A molecular link between SR protein dephosphorylation and mRNA export. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Helena Santos-Rosa,et al.  Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores , 1998, Molecular and Cellular Biology.

[24]  Z. Dominski,et al.  Inactivation of splicing factors in HeLa cells subjected to heat shock. , 1990, The Journal of biological chemistry.

[25]  T. Miki,et al.  Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs , 2007, Nucleic acids research.

[26]  C. Jolly,et al.  Optimization of Nuclear Transcript Detection by FISH and Combination with Fluorescence Immunocytochemical Detection of Transcription Factors , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[27]  S. Lindquist,et al.  The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader , 1985, Cell.

[28]  Barry Honig,et al.  GRASP2: visualization, surface properties, and electrostatics of macromolecular structures and sequences. , 2003, Methods in enzymology.

[29]  J. M. Sierra,et al.  Internal ribosome entry site drives cap-independent translation of reaper and heat shock protein 70 mRNAs in Drosophila embryos. , 2004, RNA.

[30]  P. Silver,et al.  Definition of global and transcript-specific mRNA export pathways in metazoans. , 2008, Genes & development.

[31]  T. Köcher,et al.  The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila , 2001, Current Biology.

[32]  E. Conti,et al.  The structure of the mRNA export factor TAP reveals a cis arrangement of a non‐canonical RNP domain and an LRR domain , 2000, The EMBO journal.

[33]  A. Aguilera Cotranscriptional mRNP assembly: from the DNA to the nuclear pore. , 2005, Current opinion in cell biology.

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

[35]  M. Ohno,et al.  ATP-Dependent Recruitment of Export Factor Aly/REF onto Intronless mRNAs by RNA Helicase UAP56 , 2007, Molecular and Cellular Biology.

[36]  A. Hopper,et al.  Regulation of mRNA export in response to stress in Saccharomyces cerevisiae. , 1996, Genes & development.

[37]  E. Conti,et al.  Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor. , 2001, Molecular cell.

[38]  P. Dejong,et al.  Cloning of a novel, anonymous gene from a megabase-range YAC and cosmid contig in the neurofibromatosis type 2/meningioma region on human chromosome 22q12. , 1993, Human molecular genetics.

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

[40]  Liam J. McGuffin,et al.  Improvement of the GenTHREADER Method for Genomic Fold Recognition , 2003, Bioinform..

[41]  R. Reed,et al.  TREX, SR proteins and export of mRNA. , 2005, Current opinion in cell biology.

[42]  E. Hurt,et al.  A versatile interaction platform on the Mex67–Mtr2 receptor creates an overlap between mRNA and ribosome export , 2008, The EMBO journal.

[43]  B. Blencowe,et al.  REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[44]  U. Bond,et al.  Heat shock but not other stress inducers leads to the disruption of a sub‐set of snRNPs and inhibition of in vitro splicing in HeLa cells. , 1988, The EMBO journal.

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

[46]  K. Horie-Inoue,et al.  Identification and functional analysis of consensus androgen response elements in human prostate cancer cells. , 2004, Biochemical and biophysical research communications.

[47]  C. Joshi,et al.  5' untranslated leader sequences of eukaryotic mRNAs encoding heat shock induced proteins. , 1995, Nucleic acids research.

[48]  J. Steitz,et al.  SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. , 2003, Molecular cell.

[49]  Ed Hurt,et al.  Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Torsten Schwede,et al.  BIOINFORMATICS Bioinformatics Advance Access published November 12, 2005 The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling , 2022 .

[51]  E. Izaurralde,et al.  NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila. , 2001, RNA.

[52]  Susanne Röther,et al.  The RNA polymerase II CTD kinase Ctk1 functions in translation elongation. , 2007, Genes & development.

[53]  Christine Guthrie,et al.  The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA. , 2004, Molecular cell.

[54]  M. Suyama,et al.  TAP (NXF1) Belongs to a Multigene Family of Putative RNA Export Factors with a Conserved Modular Architecture , 2000, Molecular and Cellular Biology.

[55]  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.

[56]  E. Hurt,et al.  Complex Formation between Tap and p15 Affects Binding to FG-repeat Nucleoporins and Nucleocytoplasmic Shuttling* , 2002, The Journal of Biological Chemistry.

[57]  S. Lindquist,et al.  RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis , 1986, Cell.

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

[59]  K Strässer,et al.  Yra1p, a conserved nuclear RNA‐binding protein, interacts directly with Mex67p and is required for mRNA export , 2000, The EMBO journal.

[60]  Hiroshi Kimura,et al.  The Interaction between Cap-binding Complex and RNA Export Factor Is Required for Intronless mRNA Export* , 2007, Journal of Biological Chemistry.

[61]  M. Scott,et al.  Translational control of protein synthesis in response to heat shock in D. melanogaster cells , 1980, Cell.

[62]  R. Evans,et al.  The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. , 2007, Genes & development.

[63]  M. Hampsey,et al.  Synchronicity: policing multiple aspects of gene expression by Ctk1. , 2007, Genes & development.

[64]  Michael R. Green,et al.  Localization of pre-mRNA splicing in mammalian nuclei , 1994, Nature.

[65]  U. Kutay,et al.  The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. , 2000, RNA.

[66]  B. Cullen,et al.  The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. , 1999, Genes & development.

[67]  Ed Hurt,et al.  Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2. , 2007, Molecular cell.

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

[69]  R. Morimoto,et al.  Intron-independent Association of Splicing Factors with Active Genes , 1999, The Journal of cell biology.

[70]  Henning Urlaub,et al.  Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment. , 2006, RNA.

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

[72]  Satoshi Shibata,et al.  Exportin-5 orthologues are functionally divergent among species , 2006, Nucleic acids research.