ALYREF mainly binds to the 5 (cid:2) and the 3 (cid:2) regions of the mRNA invivo

The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous invitrostudies, ALYREF binds to a region near the 5 (cid:2) end of the mRNA in a CBP80-dependent man-ner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3 (cid:2) end of the mRNA. Fur-thermore, the 3 (cid:2) processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5 (cid:2) and the 3 (cid:2) regions of the mRNA invivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3 (cid:2) processing.

[1]  P. Björk,et al.  Integration of mRNP formation and export , 2017, Cellular and Molecular Life Sciences.

[2]  Stuart A. Wilson,et al.  The role of TREX in gene expression and disease , 2016, The Biochemical journal.

[3]  K. Hofmann,et al.  A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs , 2016, Nucleic acids research.

[4]  Alexander F. Palazzo,et al.  Splicing promotes the nuclear export of β-globin mRNA by overcoming nuclear retention elements , 2015, RNA.

[5]  Rohit Reja,et al.  The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression , 2015, Cell.

[6]  Bruce L. Miller,et al.  GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport , 2015, Nature.

[7]  B. V. van Bon,et al.  THOC2 Mutations Implicate mRNA-Export Pathway in X-Linked Intellectual Disability. , 2015, American journal of human genetics.

[8]  R. Reed,et al.  Premature termination codons are recognized in the nucleus in a reading-frame-dependent manner , 2015, Cell Discovery.

[9]  Alexander F. Palazzo,et al.  The Consensus 5' Splice Site Motif Inhibits mRNA Nuclear Export , 2015, PloS one.

[10]  J. Katahira.  [Formation and nuclear export of messenger ribonucleoprotein complexes]. , 2015, Seikagaku. The Journal of Japanese Biochemical Society.

[11]  A. Williamson,et al.  THOC5 controls 3′end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100) , 2014, Nucleic acids research.

[12]  Johannes Söding,et al.  Transcriptome maps of mRNP biogenesis factors define pre-mRNA recognition. , 2014, Molecular cell.

[13]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[14]  R. Parker,et al.  Principles and properties of eukaryotic mRNPs. , 2014, Molecular cell.

[15]  Guohui Li,et al.  A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18 , 2014, Nucleic acids research.

[16]  Julian König,et al.  Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L , 2014, RNA biology.

[17]  K. Hochedlinger,et al.  The THO complex regulates pluripotency gene mRNA export and controls embryonic stem cell self-renewal and somatic cell reprogramming. , 2013, Cell stem cell.

[18]  E. O'Duibhir,et al.  Recruitment of TREX to the Transcription Machinery by Its Direct Binding to the Phospho-CTD of RNA Polymerase II , 2013, PLoS genetics.

[19]  J. Yates,et al.  Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3′ processing , 2013, RNA.

[20]  Y. Ohkawa,et al.  Human TREX component Thoc5 affects alternative polyadenylation site choice by recruiting mammalian cleavage factor I , 2013, Nucleic acids research.

[21]  Julian König,et al.  Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements , 2013, Cell.

[22]  S. Gygi,et al.  Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export , 2012, Nucleic acids research.

[23]  M. Tan,et al.  Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA , 2012, Nucleic acids research.

[24]  Stuart A. Wilson,et al.  TREX exposes the RNA binding domain of Nxf1 to enable mRNA export , 2012, Nature Communications.

[25]  R. Reed,et al.  Export and stability of naturally intronless mRNAs require specific coding region sequences and the TREX mRNA export complex , 2011, Proceedings of the National Academy of Sciences.

[26]  Blaz Zupan,et al.  iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution , 2011, Journal of visualized experiments : JoVE.

[27]  R. Luna,et al.  Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers , 2011, BMC Cancer.

[28]  J. Ule,et al.  iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution , 2010, Nature Structural &Molecular Biology.

[29]  Stuart A. Wilson,et al.  Arginine methylation of REF/ALY promotes efficient handover of mRNA to TAP/NXF1 , 2010, Nucleic acids research.

[30]  S. Cardinale,et al.  Mammalian pre-mRNA 3' end processing factor CF I m 68 functions in mRNA export. , 2009, Molecular biology of the cell.

[31]  D. Bentley,et al.  Cotranscriptional recruitment of the mRNA export factor Yra1 by direct interaction with the 3' end processing factor Pcf11. , 2009, Molecular cell.

[32]  R. Reed,et al.  Splicing promotes rapid and efficient mRNA export in mammalian cells , 2008, Proceedings of the National Academy of Sciences.

[33]  T. Rapoport,et al.  The Signal Sequence Coding Region Promotes Nuclear Export of mRNA , 2007, PLoS biology.

[34]  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.

[35]  E. Wentzel,et al.  A Hexanucleotide Element Directs MicroRNA Nuclear Import , 2007, Science.

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

[37]  G. Panayotou,et al.  RNA-binding Motif Protein 15 Binds to the RNA Transport Element RTE and Provides a Direct Link to the NXF1 Export Pathway* , 2006, Journal of Biological Chemistry.

[38]  Stuart A. Wilson,et al.  The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA. , 2006, RNA.

[39]  Xiaoling Wang,et al.  Thoc1/Hpr1/p84 Is Essential for Early Embryonic Development in the Mouse , 2006, Molecular and Cellular Biology.

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

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

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

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

[44]  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.

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

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

[47]  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.

[48]  E. Izaurralde,et al.  TAP binds to the constitutive transport element (CTE) through a novel RNA‐binding motif that is sufficient to promote CTE‐dependent RNA export from the nucleus , 1999, The EMBO journal.

[49]  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.