Yeast Shuttling SR Proteins Npl3p, Gbp2p, and Hrb1p Are Part of the Translating mRNPs, and Npl3p Can Function as a Translational Repressor

ABSTRACT A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.

[1]  X. P. Shi,et al.  Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein. , 1997, Biological signals.

[2]  A. Gingras,et al.  eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. , 1999, Annual review of biochemistry.

[3]  C. Cole,et al.  Dbp5p/Rat8p is a yeast nuclear pore‐associated DEAD‐box protein essential for RNA export , 1998, The EMBO journal.

[4]  C. Cole,et al.  An early function during transcription for the yeast mRNA export factor Dbp5p/Rat8p suggested by its genetic and physical interactions with transcription factor IIH components. , 2003, Molecular biology of the cell.

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

[6]  R. Stauber,et al.  The Human Poly(A)-binding Protein 1 Shuttles between the Nucleus and the Cytoplasm* , 1998, The Journal of Biological Chemistry.

[7]  A. Sachs,et al.  Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[8]  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.

[9]  J. Manley,et al.  Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. , 1997, Genes & development.

[10]  W. Gilbert,et al.  Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation. , 2001, RNA.

[11]  Jing Zhao,et al.  Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis , 1999, Microbiology and Molecular Biology Reviews.

[12]  E. Craig,et al.  The Yeast hsp70 Homologue Ssa Is Required for Translation and Interacts with Sis1 and Pab1 on Translating Ribosomes* , 2001, The Journal of Biological Chemistry.

[13]  P. Silver,et al.  Protein and RNA export from the nucleus. , 2002, Developmental cell.

[14]  X. Fu,et al.  Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4. , 2001, RNA.

[15]  E. Izaurralde,et al.  The interplay of nuclear mRNP assembly, mRNA surveillance and export. , 2003, Trends in cell biology.

[16]  Fred Winston,et al.  Construction of a set of convenient saccharomyces cerevisiae strains that are isogenic to S288C , 1995, Yeast.

[17]  R. Benne,et al.  Purification and characterization of protein synthesis initiation factors eIF-1, eIF-4C, eIF-4D, and eIF-5 from rabbit reticulocytes. , 1978, The Journal of biological chemistry.

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

[19]  Daniel Zenklusen,et al.  The Yeast hnRNP-Like Proteins Yra1p and Yra2p Participate in mRNA Export through Interaction with Mex67p , 2001, Molecular and Cellular Biology.

[20]  Yan Liu,et al.  Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export , 1998, The EMBO journal.

[21]  C. Cole,et al.  A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes , 1995, The Journal of cell biology.

[22]  C. Guthrie,et al.  A putative ubiquitin ligase required for efficient mRNA export differentially affects hnRNP transport , 2000, Current Biology.

[23]  J. Aitchison,et al.  Kap104p-mediated Nuclear Import , 1999, The Journal of Biological Chemistry.

[24]  J. Hauber,et al.  Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

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

[26]  A. Xu,et al.  Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display. , 2004, The Biochemical journal.

[27]  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.

[28]  C. Guthrie,et al.  Conservation in budding yeast of a kinase specific for SR splicing factors. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Sachs,et al.  Association of the yeast poly(A) tail binding protein with translation initiation factor eIF‐4G. , 1996, The EMBO journal.

[30]  M. Ruiz-Echevarría,et al.  The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay. , 2000, Molecular cell.

[31]  P. Silver,et al.  Messenger RNAs are recruited for nuclear export during transcription. , 2001, Genes & development.

[32]  B. Daneholt,et al.  A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export , 1996, The Journal of cell biology.

[33]  David Tollervey,et al.  Yeast Nop15p is an RNA‐binding protein required for pre‐rRNA processing and cytokinesis , 2003, The EMBO journal.

[34]  P. Silver,et al.  Arginine methylation facilitates the nuclear export of hnRNP proteins. , 1998, Genes & development.

[35]  U. Kutay,et al.  Nucleocytoplasmic transport: taking an inventory , 2003, Cellular and Molecular Life Sciences CMLS.

[36]  Xiaodong Cheng,et al.  Nab2p Is Required for Poly(A) RNA Export in Saccharomyces cerevisiae and Is Regulated by Arginine Methylation via Hmt1p* , 2002, The Journal of Biological Chemistry.

[37]  N. Gray,et al.  A novel role for shuttling SR proteins in mRNA translation. , 2004, Genes & development.

[38]  A. Jacobson,et al.  A single amino acid substitution in yeast eIF‐5A results in mRNA stabilization , 1998, The EMBO journal.

[39]  M. Hammarskjöld,et al.  Tap and NXT promote translation of unspliced mRNA. , 2003, Genes & development.

[40]  P. Silver,et al.  Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export. , 1999, Genes & development.

[41]  Matthias Mann,et al.  Mtr10p functions as a nuclear import receptor for the mRNA‐binding protein Npl3p , 1998, The EMBO journal.

[42]  H. Krebber,et al.  Identification of Gbp2 as a novel poly(A)+ RNA‐binding protein involved in the cytoplasmic delivery of messenger RNAs in yeast , 2003, EMBO reports.

[43]  D. Sabatini,et al.  Dissociation of mammalian polyribosomes into subunits by puromycin. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[44]  P. Silver,et al.  Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling. , 2002, Genetics.

[45]  P. Silver,et al.  A protein that shuttles between the nucleus and the cytoplasm is an important mediator of RNA export. , 1996, Genes & development.

[46]  S. Peltz,et al.  The role of Upf proteins in modulating the translation read‐through of nonsense‐containing transcripts , 2001, The EMBO journal.

[47]  M. Pool,et al.  Scp160p, an RNA-binding, Polysome-associated Protein, Localizes to the Endoplasmic Reticulum of Saccharomyces cerevisiae in a Microtubule-dependent Manner* , 2001, The Journal of Biological Chemistry.

[48]  Lionel Minvielle-Sebastia,et al.  Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export , 2002, The EMBO journal.

[49]  P. Silver,et al.  7The Yeast mRNA-binding Protein Npl3p Interacts with the Cap-binding Complex* , 2000, The Journal of Biological Chemistry.

[50]  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.

[51]  M. H. Park,et al.  Hypusine is essential for eukaryotic cell proliferation. , 1993, Biological signals.

[52]  Xiang-Dong Fu,et al.  Conserved Sr Protein Kinase Functions in Nuclear Import and Its Action Is Counteracted by Arginine Methylation in Saccharomyces cerevisiae , 2000, The Journal of cell biology.

[53]  J. Hershey,et al.  Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. , 1994, The Journal of biological chemistry.

[54]  H. Krebber,et al.  Differential Export Requirements for Shuttling Serine/Arginine-type mRNA-binding Proteins* , 2004, Journal of Biological Chemistry.

[55]  W. Merrick,et al.  Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta. , 1976, The Journal of biological chemistry.