Association of the Breast Cancer Protein MLN51 with the Exon Junction Complex via Its Speckle Localizer and RNA Binding Module*

MLN51 is a nucleocytoplasmic shuttling protein that is overexpressed in breast cancer. The function of MLN51 in mammals remains elusive. Its fly homolog, named barentsz, as well as the proteins mago nashi and tsunagi have been shown to be required for proper oskar mRNA localization to the posterior pole of the oocyte. Magoh and Y14, the human homologs of mago nashi and tsunagi, are core components of the exon junction complex (EJC). The EJC is assembled on spliced mRNAs and plays important roles in post-splicing events including mRNA export, nonsense-mediated mRNA decay, and translation. In the present study, we show that human MLN51 is an RNA-binding protein present in ribonucleo-protein complexes. By co-immunoprecipitation assays, endogenous MLN51 protein is found to be associated with EJC components, including Magoh, Y14, and NFX1/TAP, and subcellular localization studies indicate that MLN51 transiently co-localizes with Magoh in nuclear speckles. Moreover, we demonstrate that MLN51 specifically associates with spliced mRNAs in co-precipitation experiments, both in the nucleus and in the cytoplasm, at the position where the EJC is deposited. Most interesting, we have identified a region within MLN51 sufficient to bind RNA, to interact with Magoh and spliced mRNA, and to address the protein to nuclear speckles. This conserved region of MLN51 was therefore named SELOR for speckle localizer and RNA binding module. Altogether our data demonstrate that MLN51 associates with EJC in the nucleus and remains stably associated with mRNA in the cytoplasm, suggesting that its overexpression might alter mRNA metabolism in cancer.

[1]  G. Dreyfuss,et al.  Messenger-RNA-binding proteins and the messages they carry , 2002, Nature Reviews Molecular Cell Biology.

[2]  Sophie G. Martin,et al.  The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen , 2003, Development.

[3]  W. Seol,et al.  Tap: a novel cellular protein that interacts with tip of herpesvirus saimiri and induces lymphocyte aggregation. , 1997, Immunity.

[4]  David L. Spector,et al.  Nuclear speckles: a model for nuclear organelles , 2003, Nature Reviews Molecular Cell Biology.

[5]  A. Krainer,et al.  Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity , 1997, The Journal of cell biology.

[6]  C. Nüsslein-Volhard,et al.  The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner , 1988, Cell.

[7]  Daniel St Johnston,et al.  The intracellular localization of messenger RNAs , 1995, Cell.

[8]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[9]  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, The EMBO journal.

[10]  U. Lindberg,et al.  Isolation of messenger ribonucleoproteins from mammalian cells. , 1974, Journal of molecular biology.

[11]  M. Ringnér,et al.  Impact of DNA amplification on gene expression patterns in breast cancer. , 2002, Cancer research.

[12]  K. Ohe,et al.  Orphan Receptor DAX-1 Is a Shuttling RNA Binding Protein Associated with Polyribosomes via mRNA , 2000, Molecular and Cellular Biology.

[13]  J. Vonesch,et al.  Lasp-1, a Novel Type of Actin-Binding Protein Accumulating in Cell Membrane Extensions , 1998, Molecular medicine.

[14]  G. Blobel,et al.  Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein , 1988, The Journal of cell biology.

[15]  H. Le Hir,et al.  Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. , 2004, Genes & development.

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

[17]  Olivier Poch,et al.  PipeAlign: a new toolkit for protein family analysis , 2003, Nucleic Acids Res..

[18]  É. Khandjian,et al.  The fragile X mental retardation protein is associated with poly(A)+ mRNA in actively translating polyribosomes. , 1997, Human molecular genetics.

[19]  J. Yong,et al.  Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. , 2000, Molecular cell.

[20]  J. Yong,et al.  The Y14 protein communicates to the cytoplasm the position of exon–exon junctions , 2001, The EMBO journal.

[21]  A. Munnich,et al.  The RNA-binding properties of SMN: deletion analysis of the zebrafish orthologue defines domains conserved in evolution. , 1999, Human molecular genetics.

[22]  J. G. Patton,et al.  An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles. , 2001, Experimental cell research.

[23]  B. Cullen,et al.  Exon junction complexes mediate the enhancing effect of splicing on mRNA expression , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Rahul C. Deo,et al.  Recognition of Polyadenylate RNA by the Poly(A)-Binding Protein , 1999, Cell.

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

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

[27]  R. Gattoni,et al.  Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family. , 1994, The EMBO journal.

[28]  Simion I. Chiosea,et al.  The spatial targeting and nuclear matrix binding domains of SRm160 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  F. Allain,et al.  Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target. , 2000, Journal of molecular biology.

[30]  S. Kozma,et al.  Oncogenic activation of the human trk proto‐oncogene by recombination with the ribosomal large subunit protein L7a. , 1990, The EMBO journal.

[31]  Y. Jan,et al.  Staufen: a common component of mRNA transport in oocytes and neurons? , 2000, Trends in cell biology.

[32]  C. Tomasetto,et al.  Metastatic Lymph Node 51, a novel nucleo-cytoplasmic protein overexpressed in breast cancer , 2002, Oncogene.

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

[34]  P. Basset,et al.  Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17. , 1995, Genomics.

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

[36]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[37]  D. Johnston,et al.  THE POLARISATION OF THE ANTERIOR-POSTERIOR AND DORSAL-VENTRAL AXES DURING DROSOPHILA OOGENESIS , 1999 .

[38]  F. V. van Eeden,et al.  Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole , 2001, The Journal of cell biology.

[39]  K. Nakai,et al.  PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. , 1999, Trends in biochemical sciences.

[40]  Ruth Lehmann,et al.  Induction of germ cell formation by oskar , 1992, Nature.

[41]  Amos Bairoch,et al.  The PROSITE database, its status in 1995 , 1996, Nucleic Acids Res..

[42]  A. Dierich,et al.  The Net Repressor Is Regulated by Nuclear Export in Response to Anisomycin, UV, and Heat Shock , 1999, Molecular and Cellular Biology.

[43]  P. Chambon,et al.  Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins. , 1992, The EMBO journal.

[44]  R. Elliott,et al.  The mammalian homologue of mago nashi encodes a serum-inducible protein. , 1998, Genomics.

[45]  N. Hecht,et al.  The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites. , 1999, Journal of cell science.

[46]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[47]  M. Kiebler,et al.  Barentsz, a New Component of the Staufen-Containing Ribonucleoprotein Particles in Mammalian Cells, Interacts with Staufen in an RNA-Dependent Manner , 2003, The Journal of Neuroscience.

[48]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

[49]  M. Mann,et al.  eIF4A3 is a novel component of the exon junction complex. , 2004, RNA.

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

[51]  O. Monni,et al.  New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays. , 2001, Cancer research.

[52]  H. Le Hir,et al.  How introns influence and enhance eukaryotic gene expression. , 2003, Trends in biochemical sciences.

[53]  Paolo Sassone-Corsi,et al.  CREM-Dependent Transcription in Male Germ Cells Controlled by a Kinesin , 2002, Science.

[54]  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.