Global Analysis of Pub1p Targets Reveals a Coordinate Control of Gene Expression through Modulation of Binding and Stability

ABSTRACT Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1Δ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1Δ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1Δ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3′ untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.

[1]  G. Reinsel,et al.  Introduction to Mathematical Statistics (4th ed.). , 1980 .

[2]  M. Ruiz-Echevarría,et al.  The RNA Binding Protein Pub1 Modulates the Stability of Transcripts Containing Upstream Open Reading Frames , 2000, Cell.

[3]  S. Peltz,et al.  mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor. , 1993, Genes & development.

[4]  J. Stévenin,et al.  The RNA-Binding Protein TIA-1 Is a Novel Mammalian Splicing Regulator Acting through Intron Sequences Adjacent to a 5′ Splice Site , 2000, Molecular and Cellular Biology.

[5]  J. Ross,et al.  Control of messenger RNA stability in higher eukaryotes. , 1996, Trends in genetics : TIG.

[6]  O. Ozier-Kalogeropoulos,et al.  A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. , 1993, Nucleic acids research.

[7]  S. Peltz,et al.  The cap-to-tail guide to mRNA turnover , 2001, Nature Reviews Molecular Cell Biology.

[8]  P. Anderson,et al.  TIA‐1 is a translational silencer that selectively regulates the expression of TNF‐α , 2000 .

[9]  M. Gorospe,et al.  Global analysis of HuR-regulated gene expression in colon cancer systems of reducing complexity. , 2004, Gene expression.

[10]  F. Sherman,et al.  3'-end-forming signals of yeast mRNA. , 1996, Trends in biochemical sciences.

[11]  Roy Parker,et al.  Nonsense-mediated mRNA decay: terminating erroneous gene expression. , 2004, Current opinion in cell biology.

[12]  Temple F. Smith,et al.  Probabilistic prediction of Saccharomyces cerevisiae mRNA 3'-processing sites. , 2002, Nucleic acids research.

[13]  A. Shyu,et al.  RNA stabilization by the AU‐rich element binding protein, HuR, an ELAV protein , 1998, The EMBO journal.

[14]  P. Philippsen,et al.  DNA of Saccharomyces cerevisiae. , 1991, Methods in enzymology.

[15]  K. Köhrer,et al.  Preparation of high molecular weight RNA. , 1991, Methods in enzymology.

[16]  G. Dreyfuss,et al.  PUB1: a major yeast poly(A)+ RNA-binding protein. , 1993, Molecular and cellular biology.

[17]  A. Jacobson,et al.  An internal open reading frame triggers nonsense‐mediated decay of the yeast SPT10 mRNA , 1999, The EMBO journal.

[18]  John R. Gilbertson,et al.  Microarray Data Mining Using Gene Ontology , 2004, MedInfo.

[19]  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.

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

[21]  C. Gissi,et al.  Untranslated regions of mRNAs , 2002, Genome Biology.

[22]  S. Peltz,et al.  A Yeast Homologue of Hsp70, Ssa1p, Regulates Turnover of the MFA2 Transcript through Its AU-Rich 3′ Untranslated Region , 2003, Molecular and Cellular Biology.

[23]  C. Guthrie,et al.  Functional specificity of shuttling hnRNPs revealed by genome-wide analysis of their RNA binding profiles. , 2005, RNA.

[24]  S. Tenenbaum,et al.  Gene expression analysis of messenger RNP complexes. , 2004, Methods in molecular biology.

[25]  S. Capaccioli,et al.  Post‐transcriptional regulation of gene expression by degradation of messenger RNAs , 2003, Journal of cellular physiology.

[26]  J. Valcárcel,et al.  The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing. , 2000, Molecular cell.

[27]  W. P. Dirksen,et al.  TIA Proteins Are Necessary but Not Sufficient for the Tissue-specific Splicing of the Myosin Phosphatase Targeting Subunit 1* , 2004, Journal of Biological Chemistry.

[28]  G. Crooks,et al.  WebLogo: a sequence logo generator. , 2004, Genome research.

[29]  H. Hieronymus,et al.  A systems view of mRNP biology. , 2004, Genes & development.

[30]  Roy Parker,et al.  Eukaryotic mRNA decapping. , 2004, Annual review of biochemistry.

[31]  John D. Storey,et al.  Precision and functional specificity in mRNA decay , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[32]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[33]  C. Ball,et al.  Saccharomyces Genome Database. , 2002, Methods in enzymology.

[34]  N. Sonenberg,et al.  Translational control of gene expression , 2000 .

[35]  J. Keene.  Posttranscriptional generation of macromolecular complexes. , 2003, Molecules and Cells.

[36]  J. McCarthy,et al.  The Relationship between Eukaryotic Translation and mRNA Stability , 1995, The Journal of Biological Chemistry.

[37]  M. Greenberg,et al.  The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. , 1989, Genes & development.

[38]  R. Parker,et al.  Mechanisms and control of mRNA decapping in Saccharomyces cerevisiae. , 2000, Annual review of biochemistry.

[39]  Marco M. Kessler,et al.  Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast. , 1997, Genes & development.

[40]  S. Peltz,et al.  Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency. , 2000, RNA.

[41]  DAVID G. KENDALL,et al.  Introduction to Mathematical Statistics , 1947, Nature.

[42]  P. Mitchell,et al.  mRNA stability in eukaryotes. , 2000, Current opinion in genetics & development.

[43]  M. Gorospe,et al.  Identification of a target RNA motif for RNA-binding protein HuR. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  J. Steitz,et al.  Identification of HuR as a protein implicated in AUUUA‐mediated mRNA decay , 1997, The EMBO journal.

[45]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.

[46]  R. Young,et al.  Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis , 1987, Molecular and cellular biology.

[47]  T. Hughes,et al.  Genome-Wide Analysis of mRNA Stability Using Transcription Inhibitors and Microarrays Reveals Posttranscriptional Control of Ribosome Biogenesis Factors , 2004, Molecular and Cellular Biology.

[48]  M. Gorospe,et al.  RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[49]  M. Magnasco,et al.  Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes. , 2003, Genome research.

[50]  A. Raghavan,et al.  Microarray-based analyses of mRNA decay in the regulation of mammalian gene expression. , 2004, Briefings in functional genomics & proteomics.

[51]  Robert J. Schaffer,et al.  Microarray Analysis of Diurnal and Circadian-Regulated Genes in Arabidopsis , 2001, Plant Cell.

[52]  Michael R. Green,et al.  Dissecting the Regulatory Circuitry of a Eukaryotic Genome , 1998, Cell.

[53]  J. Hesketh,et al.  3' untranslated regions of c-myc and c-fos mRNAs: multifunctional elements regulating mRNA translation, degradation and subcellular localization. , 1997, Progress in molecular and subcellular biology.

[54]  Robert V. Hogg,et al.  Introduction to Mathematical Statistics. , 1966 .

[55]  M. Swanson,et al.  PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae , 1993, Molecular and cellular biology.

[56]  J. Pérez-Ortín,et al.  Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. , 2004, Molecular cell.

[57]  S. Tenenbaum,et al.  Eukaryotic mRNPs may represent posttranscriptional operons. , 2002, Molecular cell.

[58]  S. Peltz,et al.  Regulated ARE-mediated mRNA decay in Saccharomyces cerevisiae. , 2001, Molecular cell.

[59]  R. Ozawa,et al.  A comprehensive two-hybrid analysis to explore the yeast protein interactome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[60]  E. Wisman,et al.  Arabidopsis microarrays identify conserved and differentially expressed genes involved in shoot growth and development from distantly related plant species. , 2003, The Plant journal : for cell and molecular biology.

[61]  A. Shyu,et al.  A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. , 1998, Nucleic acids research.

[62]  J. A. Steitz,et al.  HuR and mRNA stability , 2001, Cellular and Molecular Life Sciences CMLS.

[63]  C. Y. Chen,et al.  Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. , 1999, Genes & development.

[64]  S. S. Houshmandi,et al.  Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast. , 2004, RNA.

[65]  M. Swanson,et al.  Control of cleavage site selection during mRNA 3′ end formation by a yeast hnRNP , 1998, The EMBO journal.

[66]  M. Karin,et al.  Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. , 1998, Science.

[67]  P. Brown,et al.  Exploring the metabolic and genetic control of gene expression on a genomic scale. , 1997, Science.

[68]  J. Malter,et al.  Cytoplasmic fate of eukaryotic mRNA: identification and characterization of AU-binding proteins. , 1997, Progress in molecular and subcellular biology.

[69]  M. Hentze,et al.  Translational repression by a complex between the iron‐responsive element of ferritin mRNA and its specific cytoplasmic binding protein is position‐dependent in vivo. , 1990, The EMBO journal.

[70]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[71]  M. Gorospe,et al.  HuR: Post-Transcriptional Paths to Malignancy , 2005, RNA biology.

[72]  S. Peltz,et al.  The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro , 1989, Molecular and cellular biology.

[73]  P. Brown,et al.  Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in Yeast , 2004, PLoS biology.