Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1

Summary Ccr4-Not is a conserved protein complex that shortens the 3′ poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates. The intact complex is more active than the exonucleases alone and has an intrinsic preference for certain RNAs. The RNA-binding protein Mmi1 is highly abundant in preparations of native Ccr4-Not. We demonstrate a high-affinity interaction between recombinant Ccr4-Not and Mmi1. Using in vitro assays, we show that Mmi1 accelerates deadenylation of target RNAs. Together, our results support a model whereby both RNA-binding proteins and the sequence context of mRNAs influence deadenylation rate to regulate gene expression.

[1]  R Parker,et al.  A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. , 1993, Genes & development.

[2]  P. Cramer,et al.  RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc 7 , 2014 .

[3]  Imre Berger,et al.  MultiBac: expanding the research toolbox for multiprotein complexes , 2011, Trends in Biochemical Sciences.

[4]  Joerg E Braun,et al.  GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets. , 2011, Molecular cell.

[5]  Jon R Lorsch,et al.  The molecular mechanics of eukaryotic translation. , 2003, Annual review of biochemistry.

[6]  R. Parker,et al.  The Transcription Factor Associated Ccr4 and Caf1 Proteins Are Components of the Major Cytoplasmic mRNA Deadenylase in Saccharomyces cerevisiae , 2001, Cell.

[7]  Chao Xu,et al.  A novel RNA-binding mode of the YTH domain reveals the mechanism for recognition of determinant of selective removal by Mmi1 , 2015, Nucleic acids research.

[8]  Liang Tong,et al.  Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain , 2014, Proceedings of the National Academy of Sciences.

[9]  W. Filipowicz,et al.  Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression. , 2014, Molecular cell.

[10]  P. Blackshear,et al.  Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by Tristetraprolin , 2013, Nature Structural &Molecular Biology.

[11]  C. Denis,et al.  The CCR4 and CAF1 Proteins of the CCR4-NOT Complex Are Physically and Functionally Separated from NOT2, NOT4, and NOT5 , 1999, Molecular and Cellular Biology.

[12]  J. Mata,et al.  A Network of Multiple Regulatory Layers Shapes Gene Expression in Fission Yeast , 2007, Molecular cell.

[13]  M. Zofall,et al.  RNA Elimination Machinery Targeting Meiotic mRNAs Promotes Facultative Heterochromatin Formation , 2012, Science.

[14]  T. Sugiyama,et al.  Red1 promotes the elimination of meiosis‐specific mRNAs in vegetatively growing fission yeast , 2011, The EMBO journal.

[15]  E. Izaurralde,et al.  A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing. , 2014, Molecular cell.

[16]  L. Van,et al.  The 1.4-Å crystal structure of the S. pombe Pop2p deadenylase subunit unveils the configuration of an active enzyme , 2007, Nucleic acids research.

[17]  Zhike Lu,et al.  m6A-dependent regulation of messenger RNA stability , 2013, Nature.

[18]  Nathan Morris,et al.  Codon Optimality Is a Major Determinant of mRNA Stability , 2015, Cell.

[19]  Nicole Cremona,et al.  A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts , 2011, Molecular biology of the cell.

[20]  L. Van,et al.  The activity and selectivity of fission yeast Pop2p are affected by a high affinity for Zn2+ and Mn2+ in the active site. , 2009, RNA.

[21]  R. Lyne,et al.  The transcriptional program of meiosis and sporulation in fission yeast , 2002, Nature Genetics.

[22]  Chuan He,et al.  RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation , 2015, Genes & development.

[23]  Ligang Wu,et al.  YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4–NOT deadenylase complex , 2016, Nature Communications.

[24]  E. Wahle,et al.  RNA decay machines: deadenylation by the Ccr4-not and Pan2-Pan3 complexes. , 2013, Biochimica et biophysica acta.

[25]  Y. Hiraoka,et al.  Selective elimination of messenger RNA prevents an incidence of untimely meiosis , 2006, Nature.

[26]  W. Filipowicz,et al.  miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs , 2011, Nature Structural &Molecular Biology.

[27]  J. Leatherwood,et al.  A Complex Gene Regulatory Mechanism that Operates at the Nexus of Multiple RNA Processing Decisions , 2009, Nature Structural &Molecular Biology.

[28]  J. Lykke-Andersen,et al.  Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. , 2005, Genes & development.

[29]  E. Wahle,et al.  Rapid ATP-dependent Deadenylation of nanos mRNA in a Cell-free System from Drosophila Embryos* , 2006, Journal of Biological Chemistry.

[30]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[31]  Jeffrey Wilusz,et al.  The highways and byways of mRNA decay , 2007, Nature Reviews Molecular Cell Biology.

[32]  Masayuki Yamamoto,et al.  Meiotic long non-coding meiRNA accumulates as a dot at its genetic locus facilitated by Mmi1 and plays as a decoy to lure Mmi1 , 2014, Open Biology.

[33]  J. Coller,et al.  The deadenylase components Not2p, Not3p, and Not5p promote mRNA decapping , 2016, RNA.

[34]  S. Stamm,et al.  YTH: a new domain in nuclear proteins. , 2002, Trends in biochemical sciences.

[35]  Geoffrey J. Barton,et al.  Jalview Version 2—a multiple sequence alignment editor and analysis workbench , 2009, Bioinform..

[36]  M. Wickens,et al.  Translational Repression by Deadenylases* , 2010, The Journal of Biological Chemistry.

[37]  Y. Saga,et al.  Interaction between NANOS2 and the CCR4-NOT Deadenylation Complex Is Essential for Male Germ Cell Development in Mouse , 2012, PloS one.

[38]  R. Stefl,et al.  Faculty Opinions recommendation of RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7. , 2014 .

[39]  G. S. Winkler,et al.  The enzyme activities of Caf1 and Ccr4 are both required for deadenylation by the human Ccr4–Not nuclease module , 2015, The Biochemical journal.

[40]  J. Lachuer,et al.  Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast , 2012, The EMBO journal.

[41]  E. Izaurralde,et al.  Structure and assembly of the NOT module of the human CCR4–NOT complex , 2013, Nature Structural &Molecular Biology.

[42]  F. Netter,et al.  Supplemental References , 2002, We Came Naked and Barefoot.

[43]  A. Heck,et al.  Human Ccr4-Not complexes contain variable deadenylase subunits. , 2009, The Biochemical journal.

[44]  Steffen Schmidt,et al.  The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4–NOT deadenylase complex , 2012, Nucleic acids research.

[45]  D. Bartel,et al.  Poly(A)-tail profiling reveals an embryonic switch in translational control , 2014, Nature.

[46]  B. Séraphin,et al.  In Vivo Targeting of the Yeast Pop2 Deadenylase Subunit to Reporter Transcripts Induces Their Rapid Degradation and Generates New Decay Intermediates* , 2006, Journal of Biological Chemistry.

[47]  E. Valkov,et al.  Distinct modes of recruitment of the CCR4–NOT complex by Drosophila and vertebrate Nanos , 2016, The EMBO journal.

[48]  B. Séraphin,et al.  Structure and RNA-binding properties of the Not1–Not2–Not5 module of the yeast Ccr4–Not complex , 2013, Nature Structural &Molecular Biology.

[49]  W. Olivas,et al.  Roles of Puf proteins in mRNA degradation and translation , 2011, Wiley interdisciplinary reviews. RNA.

[50]  C. Yun,et al.  A novel protein, Pho92, has a conserved YTH domain and regulates phosphate metabolism by decreasing the mRNA stability of PHO4 in Saccharomyces cerevisiae. , 2014, The Biochemical journal.

[51]  Michael Hiller,et al.  The YTH Domain Is a Novel RNA Binding Domain* , 2010, The Journal of Biological Chemistry.

[52]  J. Mata,et al.  Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast , 2015, Epigenetics & Chromatin.

[53]  Y. Hiraoka,et al.  Hexanucleotide motifs mediate recruitment of the RNA elimination machinery to silent meiotic genes , 2012, Open Biology.

[54]  M. Wickens,et al.  PUF proteins bind Pop2p to regulate messenger RNAs , 2006, Nature Structural &Molecular Biology.

[55]  L. Vasiljeva,et al.  Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1 , 2015, Cell reports.

[56]  Peter F. Stadler,et al.  ViennaRNA Package 2.0 , 2011, Algorithms for Molecular Biology.

[57]  Toru Suzuki,et al.  Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity , 2010, The EMBO journal.

[58]  Kevin Struhl,et al.  Global Analysis of mRNA Isoform Half-Lives Reveals Stabilizing and Destabilizing Elements in Yeast , 2014, Cell.

[59]  N. Sonenberg,et al.  miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4–NOT , 2011, Nature Structural &Molecular Biology.

[60]  T. Preiss,et al.  Widespread use of poly(A) tail length control to accentuate expression of the yeast transcriptome. , 2007, RNA.

[61]  M. Collart.  The Ccr4‐Not complex is a key regulator of eukaryotic gene expression , 2016, Wiley interdisciplinary reviews. RNA.

[62]  M. Wickens,et al.  Two Yeast PUF Proteins Negatively Regulate a Single mRNA* , 2007, Journal of Biological Chemistry.

[63]  Felice-Alessio Bava,et al.  Translational control by changes in poly(A) tail length: recycling mRNAs , 2012, Nature Structural &Molecular Biology.

[64]  Joanna M. Kasprzak,et al.  The architecture of the Schizosaccharomyces pombe CCR4-NOT complex , 2016, Nature Communications.

[65]  B. Séraphin,et al.  Architecture of the nuclease module of the yeast Ccr4-not complex: the Not1-Caf1-Ccr4 interaction. , 2012, Molecular cell.

[66]  M. Wickens,et al.  Multifunctional deadenylase complexes diversify mRNA control , 2008, Nature Reviews Molecular Cell Biology.

[67]  M. Wickens,et al.  PUF Protein-mediated Deadenylation Is Catalyzed by Ccr4p* , 2007, Journal of Biological Chemistry.

[68]  E. Izaurralde,et al.  Structural basis for the Nanos-mediated recruitment of the CCR4–NOT complex and translational repression , 2014, Genes & development.

[69]  E. Conti,et al.  Architecture of the Ubiquitylation Module of the Yeast Ccr4-Not Complex , 2015, Structure.