hUPF2 Silencing Identifies Physiologic Substrates of Mammalian Nonsense-Mediated mRNA Decay

ABSTRACT Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that selectively degrades aberrant mRNAs with premature termination codons (PTCs). Although a small number of cases exist in mammals, where NMD controls levels of physiologic PTC transcripts, it is still unclear whether the engagement of NMD in posttranscriptional control of gene expression is a more prevalent phenomenon. To identify physiologic NMD substrates and to study how NMD silencing affects the overall dynamics of a cell, we stably down-regulated hUPF2, the human homolog of the yeast NMD factor UPF2, by RNA interference. As expected, hUPF2-silenced HeLa cells were impaired in their ability to recognize ectopically expressed aberrant PTC transcripts. Surprisingly, hUPF2 silencing did not affect cell growth and viability but clearly diminished phosphorylation of hUPF1, suggesting a role of hUPF2 in modulating NMD activity through phosphorylation of hUPF1. Genome-wide DNA microarray expression profiling identified 37 novel up-regulated and 57 down-regulated transcripts in hUPF2-silenced cells. About 60% of the up-regulated mRNAs carry typical NMD motifs. Hence, NMD is important not only for maintaining the transcriptome integrity by removing nonfunctional and aberrant PTC-bearing transcripts but also for posttranscriptional control of selected physiologic transcripts with NMD features.

[1]  Alexandra Paillusson,et al.  A GFP-based reporter system to monitor nonsense-mediated mRNA decay , 2005, Nucleic acids research.

[2]  Luc DesGroseillers,et al.  Mammalian Staufen1 Recruits Upf1 to Specific mRNA 3′UTRs so as to Elicit mRNA Decay , 2005, Cell.

[3]  Francisco Martinez-Murillo,et al.  Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise , 2004, Nature Genetics.

[4]  P. Anderson,et al.  SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans , 2004, Molecular and Cellular Biology.

[5]  M. Malim,et al.  APOBEC-Mediated Editing of Viral RNA , 2004, Science.

[6]  L. Maquat.  Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics , 2004, Nature Reviews Molecular Cell Biology.

[7]  C. Gooding,et al.  Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay. , 2004, Molecular cell.

[8]  S. Brenner,et al.  An unappreciated role for RNA surveillance , 2004, Genome Biology.

[9]  Shuyun Dong,et al.  Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5' to 3' mRNA decay pathways in yeast. , 2003, Molecular cell.

[10]  K. Anders,et al.  Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7. , 2003, Molecular cell.

[11]  H. Jäck,et al.  Interaction of Murine Precursor B Cell Receptor with Stroma Cells Is Controlled by the Unique Tail of λ5 and Stroma Cell-Associated Heparan Sulfate 1 , 2003, The Journal of Immunology.

[12]  Robert H. Silverman,et al.  Activation of the interferon system by short-interfering RNAs , 2003, Nature Cell Biology.

[13]  P. Bork,et al.  Nonsense‐mediated mRNA decay in Drosophila:at the intersection of the yeast and mammalian pathways , 2003, The EMBO journal.

[14]  M. Hentze,et al.  Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells. , 2003, The Biochemical journal.

[15]  R. Iggo,et al.  Induction of an interferon response by RNAi vectors in mammalian cells , 2003, Nature Genetics.

[16]  T. Nazarenus,et al.  The Upf-dependent decay of wild-type PPR1 mRNA depends on its 5'-UTR and first 92 ORF nucleotides. , 2003, Nucleic acids research.

[17]  M. Culbertson,et al.  Looking at mRNA decay pathways through the window of molecular evolution. , 2003, Current opinion in genetics & development.

[18]  Y. Shiloh.  ATM and related protein kinases: safeguarding genome integrity , 2003, Nature Reviews Cancer.

[19]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[20]  K. Anders,et al.  SMG‐5, required for C.elegans nonsense‐mediated mRNA decay, associates with SMG‐2 and protein phosphatase 2A , 2003, The EMBO journal.

[21]  E. Schuetz,et al.  Nonsense mediated decay downregulates conserved alternatively spliced ABCC4 transcripts bearing nonsense codons. , 2003, Human molecular genetics.

[22]  L. Maquat,et al.  Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1. , 2003, RNA.

[23]  S. Brenner,et al.  Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Mendell,et al.  Separable Roles for rent1/hUpf1 in Altered Splicing and Decay of Nonsense Transcripts , 2002, Science.

[25]  G. Dreyfuss,et al.  Translation Is Required to Remove Y14 from mRNAs in the Cytoplasm , 2002, Current Biology.

[26]  L. Maquat,et al.  The exon junction complex is detected on CBP80‐bound but not eIF4E‐bound mRNA in mammalian cells: dynamics of mRNP remodeling , 2002, The EMBO journal.

[27]  Jun Wang,et al.  A Quality Control Pathway That Down-regulates Aberrant T-cell Receptor (TCR) Transcripts by a Mechanism Requiring UPF2 and Translation* , 2002, The Journal of Biological Chemistry.

[28]  L. Carastro,et al.  Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta. , 2002, Nucleic acids research.

[29]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[30]  G. Dreyfuss,et al.  Role of the Nonsense-Mediated Decay Factor hUpf3 in the Splicing-Dependent Exon-Exon Junction Complex , 2001, Science.

[31]  L. Maquat,et al.  Evidence for a Pioneer Round of mRNA Translation mRNAs Subject to Nonsense-Mediated Decay in Mammalian Cells Are Bound by CBP80 and CBP20 , 2001, Cell.

[32]  Y. Taya,et al.  Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay. , 2001, Genes & development.

[33]  L. Maquat,et al.  Cloning of a Novel Phosphatidylinositol Kinase-related Kinase , 2001, The Journal of Biological Chemistry.

[34]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[35]  A Sureau,et al.  SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs , 2001, The EMBO journal.

[36]  J. Mendell,et al.  Rent1, a trans-effector of nonsense-mediated mRNA decay, is essential for mammalian embryonic viability. , 2001, Human molecular genetics.

[37]  L. Maquat,et al.  Identification and Characterization of Human Orthologues to Saccharomyces cerevisiae Upf2 Protein and Upf3 Protein (Caenorhabditis elegans SMG-4) , 2001, Molecular and Cellular Biology.

[38]  L. Maquat,et al.  Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. , 2001, RNA.

[39]  J. Steitz,et al.  Human Upf Proteins Target an mRNA for Nonsense-Mediated Decay When Bound Downstream of a Termination Codon , 2000, Cell.

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

[41]  J. Mendell,et al.  Novel Upf2p Orthologues Suggest a Functional Link between Translation Initiation and Nonsense Surveillance Complexes , 2000, Molecular and Cellular Biology.

[42]  Q. Mitrovich,et al.  Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans. , 2000, Genes & development.

[43]  S. Peltz,et al.  Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay. , 2000, RNA.

[44]  H. Le Hir,et al.  Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. , 2000, Genes & development.

[45]  M. Culbertson,et al.  Yeast Upf Proteins Required for RNA Surveillance Affect Global Expression of the Yeast Transcriptome , 1999, Molecular and Cellular Biology.

[46]  K. Anders,et al.  SMG-2 Is a Phosphorylated Protein Required for mRNA Surveillance in Caenorhabditis elegans and Related to Upf1p of Yeast , 1999, Molecular and Cellular Biology.

[47]  H. Koeffler,et al.  Molecular Cloning of Transferrin Receptor 2 , 1999, The Journal of Biological Chemistry.

[48]  G. M. Wilson,et al.  Regulation of AUF1 Expression via Conserved Alternatively Spliced Elements in the 3′ Untranslated Region , 1999, Molecular and Cellular Biology.

[49]  L. Maquat,et al.  A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[50]  M. Hentze,et al.  Binary specification of nonsense codons by splicing and cytoplasmic translation , 1998, The EMBO journal.

[51]  L. Maquat,et al.  A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. , 1998, Trends in biochemical sciences.

[52]  L. Maquat,et al.  Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA , 1998, Molecular and Cellular Biology.

[53]  Shulin Li,et al.  Nonsense surveillance in lymphocytes? , 1998, Immunity.

[54]  F. Grosveld,et al.  Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients. , 1998, Science.

[55]  M J Clemens,et al.  The double-stranded RNA-dependent protein kinase PKR: structure and function. , 1997, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[56]  H. Jäck,et al.  Cloning and characterization of HUPF1, a human homolog of the Saccharomyces cerevisiae nonsense mRNA-reducing UPF1 protein. , 1997, Nucleic acids research.

[57]  S. Peltz,et al.  Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover , 1996, Molecular and cellular biology.

[58]  F. Spencer,et al.  Mammalian orthologues of a yeast regulator of nonsense transcript stability. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[59]  L. Maquat.  When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. , 1995, RNA.

[60]  John Calvin Reed,et al.  Cloning and functional analysis of BAG-1: A novel Bcl-2-binding protein with anti-cell death activity , 1995, Cell.

[61]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[62]  M. Culbertson,et al.  Gene products that promote mRNA turnover in Saccharomyces cerevisiae , 1992, Molecular and cellular biology.

[63]  P. Slonimski,et al.  NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. , 1992, Journal of molecular biology.

[64]  A. Krainer,et al.  Functional expression of cloned human splicing factor SF2: homology to rna-binding proteins, U1 70K, and drosophila splicing regulators , 1991, Cell.

[65]  V. Ambros,et al.  A new kind of informational suppression in the nematode Caenorhabditis elegans. , 1989, Genetics.

[66]  M. Wabl,et al.  Translation affects immunoglobulin mRNA stability , 1989, European journal of immunology.

[67]  M. Wabl,et al.  Immunoglobulin mRNA stability varies during B lymphocyte differentiation. , 1988, The EMBO journal.

[68]  M. Potash,et al.  Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. , 1985, The EMBO journal.

[69]  S. Orkin,et al.  Nonsense and frameshift mutations in beta 0-thalassemia detected in cloned beta-globin genes. , 1981, The Journal of biological chemistry.

[70]  R. Losson,et al.  Interference of nonsense mutations with eukaryotic messenger RNA stability. , 1979, Proceedings of the National Academy of Sciences of the United States of America.