A Yeast Exosome Cofactor, Mpp6, Functions in RNA Surveillance and in the Degradation of Noncoding RNA Transcripts

ABSTRACT A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 3′→5′ exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6Δ were confirmed for rrp47Δ and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of “cryptic” noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism.

[1]  M. Fromont-Racine,et al.  Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles , 2008, Proceedings of the National Academy of Sciences.

[2]  D. Tollervey,et al.  The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3′ processing pathway , 2008, Nucleic acids research.

[3]  D. Tollervey,et al.  The nuclear RNA surveillance machinery: the link between ncRNAs and genome structure in budding yeast? , 2008, Biochimica et biophysica acta.

[4]  A. Morillon,et al.  A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in S. cerevisiae. , 2008, Genes & development.

[5]  S. Buratowski,et al.  Transcription termination and RNA degradation contribute to silencing of RNA polymerase II transcription within heterochromatin. , 2008, Molecular cell.

[6]  J. Bähler,et al.  Global Role for Polyadenylation-Assisted Nuclear RNA Degradation in Posttranscriptional Gene Silencing , 2007, Molecular and Cellular Biology.

[7]  D. Tollervey,et al.  Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control , 2007, The EMBO journal.

[8]  D. Tollervey,et al.  Roles of the HEAT repeat proteins Utp10 and Utp20 in 40S ribosome maturation. , 2007, RNA.

[9]  P. Mitchell,et al.  The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein , 2007, Nucleic acids research.

[10]  Howard Y. Chang,et al.  Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs , 2007, Cell.

[11]  William Stafford Noble,et al.  Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project , 2007, Nature.

[12]  Steven P. Gygi,et al.  RNAi-Dependent and -Independent RNA Turnover Mechanisms Contribute to Heterochromatic Gene Silencing , 2007, Cell.

[13]  Rune Thomsen,et al.  Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants , 2007, The EMBO journal.

[14]  K. Struhl.  Transcriptional noise and the fidelity of initiation by RNA polymerase II , 2007, Nature Structural &Molecular Biology.

[15]  Christopher L. Warren,et al.  Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase. , 2006, Molecular cell.

[16]  J. Corden,et al.  Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. , 2006, Molecular cell.

[17]  David Tollervey,et al.  RNA-quality control by the exosome , 2006, Nature Reviews Molecular Cell Biology.

[18]  D. Tollervey,et al.  Surveillance of nuclear‐restricted pre‐ribosomes within a subnucleolar region of Saccharomyces cerevisiae , 2006, The EMBO journal.

[19]  J. Rain,et al.  A functional network involved in the recycling of nucleocytoplasmic pre-60S factors , 2006, The Journal of cell biology.

[20]  D. Tollervey,et al.  Yeast Trf5p is a nuclear poly(A) polymerase , 2006, EMBO reports.

[21]  S. Buratowski,et al.  Nrd1 interacts with the nuclear exosome for 3' processing of RNA polymerase II transcripts. , 2006, Molecular cell.

[22]  G. Pruijn,et al.  MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation , 2005, Nucleic acids research.

[23]  B. Séraphin,et al.  Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase , 2005, Cell.

[24]  E. Petfalski,et al.  RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex , 2005, Cell.

[25]  W. Keller,et al.  A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control , 2005, PLoS biology.

[26]  T. Jensen,et al.  A Link between Transcription and mRNP Quality in Saccharomyces cerevisiae , 2005, RNA biology.

[27]  J. Rehfeld,et al.  Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae. , 2004, European journal of biochemistry.

[28]  David Tollervey,et al.  A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits. , 2004, Genes & development.

[29]  E. O’Shea,et al.  Global analysis of protein expression in yeast , 2003, Nature.

[30]  M. Mann,et al.  Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs , 2003, Molecular and Cellular Biology.

[31]  Brendan J. Frey,et al.  A Panoramic View of Yeast Noncoding RNA Processing , 2003, Cell.

[32]  M. Mann,et al.  Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. , 2003, Analytical chemistry.

[33]  Juri Rappsilber,et al.  Microcolumns with self-assembled particle frits for proteomics. , 2002, Journal of chromatography. A.

[34]  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.

[35]  M. Rosbash,et al.  Interactions between mRNA Export Commitment, 3′-End Quality Control, and Nuclear Degradation , 2002, Molecular and Cellular Biology.

[36]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[37]  D. Tollervey,et al.  Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs. , 2002, Molecular cell.

[38]  D. Tollervey,et al.  Ssf1p prevents premature processing of an early pre-60S ribosomal particle. , 2002, Molecular cell.

[39]  D. Brow,et al.  RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts , 2001, Nature.

[40]  Y. Araki,et al.  Ski7p G protein interacts with the exosome and the Ski complex for 3′‐to‐5′ mRNA decay in yeast , 2001, The EMBO journal.

[41]  K. Wu,et al.  Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae. , 2001, Nucleic acids research.

[42]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[43]  Trevor Lithgow,et al.  A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae , 2000, The EMBO journal.

[44]  R. Baker,et al.  Function of the Ski4p (Csl4p) and Ski7p Proteins in 3′-to-5′ Degradation of mRNA , 2000, Molecular and Cellular Biology.

[45]  D. Tollervey,et al.  Identification of a Regulated Pathway for Nuclear Pre-mRNA Turnover , 2000, Cell.

[46]  P. Mitchell,et al.  Degradation of ribosomal RNA precursors by the exosome. , 2000, Nucleic acids research.

[47]  R. Parker,et al.  Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast , 2000, The EMBO journal.

[48]  M. Swanson,et al.  A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II. , 2000, Genetics.

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

[50]  A. Podtelejnikov,et al.  The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases. , 1999, Genes & development.

[51]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[52]  D. Tollervey,et al.  RRP5 is required for formation of both 18S and 5.8S rRNA in yeast. , 1996, The EMBO journal.

[53]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[54]  G. Blobel,et al.  Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins , 1995, Cell.

[55]  R. Schneiter,et al.  Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae. , 1995, Molecular biology of the cell.

[56]  L. Minvielle-Sebastia,et al.  RNA14 and RNA15 proteins as components of a yeast pre-mRNA 3'-end processing factor. , 1994, Science.

[57]  M. Plumpton,et al.  A dominant negative mutation in the conserved RNA helicase motif ‘SAT’ causes splicing factor PRP2 to stall in spliceosomes. , 1994, The EMBO journal.

[58]  M. Plumpton,et al.  The splicing factor PRP2, a putative RNA helicase, interacts directly with pre‐mRNA. , 1994, The EMBO journal.

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

[60]  D. Tollervey,et al.  Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U‐snRNPs. , 1987, The EMBO journal.

[61]  G. Chanfreau,et al.  Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome. , 2006, RNA.

[62]  A. Podtelejnikov,et al.  Rrp47p Is an Exosome-Associated Protein Required for the 3 (cid:2) Processing of Stable RNAs , 2003 .

[63]  D. Tollervey,et al.  Ribosome synthesis in Saccharomyces cerevisiae. , 1999, Annual review of genetics.