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Roles of mRNA Fate Modulators Dhh1 and Pat1 in TNRC6-dependent Gene Silencing Recapitulated in Yeast*

  • Shiho Makino
    Footnotes
    Affiliations
    From the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan, the
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  • Yuichiro Mishima
    Correspondence
    To whom correspondence may be addressed: Dept. of Medical Genome Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. Tel.: 81-3-5841-7894; Fax: 81-3-5841-8485;
    Affiliations
    Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

    Department of Medical Genome Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan, and the
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  • Kunio Inoue
    Affiliations
    Graduate School of Science, Kobe University, Kobe 657-8501, Japan
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  • Toshifumi Inada
    Correspondence
    To whom correspondence may be addressed: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan. Tel.: 81-22-795-6874; Fax: 81-22-795-6873; E-mail:
    Affiliations
    From the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan, the
    Search for articles by this author
  • Author Footnotes
    * This study was supported by Grant-in-aid for Scientific Research on Innovative Areas “RNA regulation” (20112006) and “Nascent chain biology” (26116003) (to T. I.) and “non-coding RNA” (24115711) (to Y. M.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Research Grants in the Natural Sciences, Mitsubishi Foundation (to T. I.).
    1 Recipient of a Japan Society for the Promotion of Science Research Fellowship.
Open AccessPublished:February 05, 2015DOI:https://doi.org/10.1074/jbc.M114.615088
      The CCR4-NOT complex, the major deadenylase in eukaryotes, plays crucial roles in gene expression at the levels of transcription, mRNA decay, and protein degradation. GW182/TNRC6 proteins, which are core components of the microRNA-induced silencing complex in animals, stimulate deadenylation and repress translation via recruitment of the CCR4-NOT complex. Here we report a heterologous experimental system that recapitulates the recruitment of CCR4-NOT complex by TNRC6 in S. cerevisiae. Using this system, we characterize conserved functions of the CCR4-NOT complex. The complex stimulates degradation of mRNA from the 5′ end by Xrn1, in a manner independent of both translation and deadenylation. This degradation pathway is probably conserved in miRNA-mediated gene silencing in zebrafish. Furthermore, the mRNA fate modulators Dhh1 and Pat1 redundantly stimulate mRNA decay, but both factors are required for poly(A) tail-independent translation repression by tethered TNRC6A. Our tethering-based reconstitution system reveals that the conserved architecture of Not1/CNOT1 provides a binding surface for TNRC6, thereby connecting microRNA-induced silencing complex to the decapping machinery as well as the translation apparatus.

      Introduction

      The CCR4-NOT complex is a multisubunit complex involved in many aspects of mRNA metabolism (
      • Wahle E.
      • Winkler G.S.
      RNA decay machines: deadenylation by the Ccr4-not and Pan2-Pan3 complexes.
      ,
      • Collart M.A.
      • Panasenko O.O.
      The Ccr4-Not complex.
      • Miller J.E.
      • Reese J.C.
      Ccr4-Not complex: the control freak of eukaryotic cells.
      ). Its conserved functions include deadenylation catalyzed by the two deadenylase subunits CAF1/POP2 (CNOT7/8 in vertebrates) and CCR4 (CNOT6 in vertebrates). These two enzymes are incorporated into the complex via a direct interaction between CAF1 and the scaffold protein CNOT1 (
      • Basquin J.
      • Roudko V.V.
      • Rode M.
      • Basquin C.
      • Séraphin B.
      • Conti E.
      Architecture of the nuclease module of the yeast Ccr4-not complex: the Not1-Caf1-Ccr4 interaction.
      ,
      • Petit A.P.
      • Wohlbold L.
      • Bawankar P.
      • Huntzinger E.
      • Schmidt S.
      • Izaurralde E.
      • Weichenrieder O.
      The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4-NOT deadenylase complex.
      ). Thus, recruitment of the CCR4-NOT complex to mRNAs promotes deadenylation, which is usually followed by decapping and 5′-to-3′ degradation by Xrn1 (
      • Parker R.
      RNA degradation in Saccharomyces cerevisae.
      ,
      • Coller J.
      • Parker R.
      Eukaryotic mRNA decapping.
      ). In addition, recent studies have shown that the CCR4-NOT complex provides a link to the decapping machinery. For example, in Saccharomyces cerevisiae, the CCR4-NOT complex associates with Dhh1, a decapping activator (
      • Maillet L.
      • Collart M.A.
      Interaction between Not1p, a component of the Ccr4-not complex, a global regulator of transcription, and Dhh1p, a putative RNA helicase.
      ). Similarly, in Drosophila and mammals, the CCR4-NOT complex interacts with the Dhh1 homolog Me31B/DDX6/RCK1/p54 and the Pat1 homolog HPat/PatL1 (
      • Ozgur S.
      • Chekulaeva M.
      • Stoecklin G.
      Human Pat1b connects deadenylation with mRNA decapping and controls the assembly of processing bodies.
      • Haas G.
      • Braun J.E.
      • Igreja C.
      • Tritschler F.
      • Nishihara T.
      • Izaurralde E.
      HPat provides a link between deadenylation and decapping in metazoa.
      ,
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ). HPat/PatL1 in turn associates with the decapping enzyme Dcp2 and its activators Dcp1 and Edc3, thereby organizing assembly of the decapping machinery (
      • Ozgur S.
      • Chekulaeva M.
      • Stoecklin G.
      Human Pat1b connects deadenylation with mRNA decapping and controls the assembly of processing bodies.
      ,
      • Tritschler F.
      • Braun J.E.
      • Motz C.
      • Igreja C.
      • Haas G.
      • Truffault V.
      • Izaurralde E.
      • Weichenrieder O.
      DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa.
      ,
      • Tritschler F.
      • Eulalio A.
      • Helms S.
      • Schmidt S.
      • Coles M.
      • Weichenrieder O.
      • Izaurralde E.
      • Truffault V.
      Similar modes of interaction enable Trailer Hitch and EDC3 to associate with DCP1 and Me31B in distinct protein complexes.
      ). Moreover, Dhh1 and Pat1 also function in translation repression. In S. cerevisiae, Dhh1 or Pat1 is required for translation repression under glucose deprivation, and both Dhh1 and Pat1 repress translation initiation in vitro (
      • Coller J.
      • Parker R.
      General translational repression by activators of mRNA decapping.
      ). Drosophila Me31B and vertebrate DDX6 also act as translation repressors (
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      ,
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ,
      • Minshall N.
      • Thom G.
      • Standart N.
      A conserved role of a DEAD box helicase in mRNA masking.
      • Nakamura A.
      • Amikura R.
      • Hanyu K.
      • Kobayashi S.
      Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis.
      ,
      • Anderson J.S.
      • Parker R.
      RNA turnover: the helicase story unwinds.
      • Minshall N.
      • Kress M.
      • Weil D.
      • Standart N.
      Role of p54 RNA helicase activity and its C-terminal domain in translational repression, P-body localization and assembly.
      ). These observations imply that the CCR4-NOT complex coordinates multiple processes of mRNA degradation and translation repression rather than merely promoting deadenylation.
      MicroRNAs (miRNAs)
      The abbreviations used are: miRNA
      microRNA
      DIG
      digoxigenin
      qRT-PCR and qPCR
      quantitative RT-PCR and PCR, respectively
      MO
      morpholino oligomer
      miRISC
      miRNA-induced silencing complex
      PABP
      poly(A)-binding protein
      cRACE
      circularized rapid amplification of cDNA ends
      DN
      dominant negative.
      are small non-coding RNAs that negatively regulate gene expression by inducing translational repression, mRNA degradation, and deadenylation (
      • Fabian M.R.
      • Sonenberg N.
      • Filipowicz W.
      Regulation of mRNA translation and stability by microRNAs.
      • Lim L.P.
      • Lau N.C.
      • Garrett-Engele P.
      • Grimson A.
      • Schelter J.M.
      • Castle J.
      • Bartel D.P.
      • Linsley P.S.
      • Johnson J.M.
      Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
      ,
      • Giraldez A.J.
      • Mishima Y.
      • Rihel J.
      • Grocock R.J.
      • Van Dongen S.
      • Inoue K.
      • Enright A.J.
      • Schier A.F.
      Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs.
      ,
      • Mishima Y.
      • Giraldez A.J.
      • Takeda Y.
      • Fujiwara T.
      • Sakamoto H.
      • Schier A.F.
      • Inoue K.
      Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430.
      ,
      • Wu L.
      • Fan J.
      • Belasco J.G.
      MicroRNAs direct rapid deadenylation of mRNA.
      ,
      • Behm-Ansmant I.
      • Rehwinkel J.
      • Doerks T.
      • Stark A.
      • Bork P.
      • Izaurralde E.
      mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
      ,
      • Eulalio A.
      • Huntzinger E.
      • Nishihara T.
      • Rehwinkel J.
      • Fauser M.
      • Izaurralde E.
      Deadenylation is a widespread effect of miRNA regulation.
      ,
      • Huntzinger E.
      • Izaurralde E.
      Gene silencing by microRNAs: contributions of translational repression and mRNA decay.
      ,
      • Rehwinkel J.
      • Behm-Ansmant I.
      • Gatfield D.
      • Izaurralde E.
      A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing.
      • Eulalio A.
      • Rehwinkel J.
      • Stricker M.
      • Huntzinger E.
      • Yang S.F.
      • Doerks T.
      • Dorner S.
      • Bork P.
      • Boutros M.
      • Izaurralde E.
      Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing.
      ). miRNAs regulate their target mRNAs by associating with specific protein factors to form the miRNA-induced silencing complex (miRISC). Argonaute (Ago), a core component of miRISC, directly incorporates miRNAs (
      • Meister G.
      Argonaute proteins: functional insights and emerging roles.
      ). Drosophila GW182 and its vertebrate ortholog TNRC6A-C (trinucleotide repeat-containing 6 A-C) interact with Ago via their N-terminal glycine and tryptophan (GW) repeats, whereas their C-terminal silencing domains provide a platform for interactions with RNA regulatory factors, including poly(A)-binding protein (PABP), PAN3 of the PAN2-3 deadenylase complex, and CNOT1 (
      • Behm-Ansmant I.
      • Rehwinkel J.
      • Doerks T.
      • Stark A.
      • Bork P.
      • Izaurralde E.
      mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
      ,
      • Eulalio A.
      • Helms S.
      • Fritzsch C.
      • Fauser M.
      • Izaurralde E.
      A C-terminal silencing domain in GW182 is essential for miRNA function.
      • Till S.
      • Lejeune E.
      • Thermann R.
      • Bortfeld M.
      • Hothorn M.
      • Enderle D.
      • Heinrich C.
      • Hentze M.W.
      • Ladurner A.G.
      A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain.
      ,
      • Fabian M.R.
      • Cieplak M.K.
      • Frank F.
      • Morita M.
      • Green J.
      • Srikumar T.
      • Nagar B.
      • Yamamoto T.
      • Raught B.
      • Duchaine T.F.
      • Sonenberg N.
      miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT.
      ,
      • Chekulaeva M.
      • Mathys H.
      • Zipprich J.T.
      • Attig J.
      • Colic M.
      • Parker R.
      • Filipowicz W.
      miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.
      ,
      • Braun J.E.
      • Huntzinger E.
      • Fauser M.
      • Izaurralde E.
      GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets.
      • Christie M.
      • Boland A.
      • Huntzinger E.
      • Weichenrieder O.
      • Izaurralde E.
      Structure of the PAN3 pseudokinase reveals the basis for interactions with the PAN2 deadenylase and the GW182 proteins.
      ). The CCR4-NOT complex, which is recruited to mRNAs by miRISC, promotes deadenylation via the activities of CAF1 and CCR4 (
      • Behm-Ansmant I.
      • Rehwinkel J.
      • Doerks T.
      • Stark A.
      • Bork P.
      • Izaurralde E.
      mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
      ,
      • Fabian M.R.
      • Mathonnet G.
      • Sundermeier T.
      • Mathys H.
      • Zipprich J.T.
      • Svitkin Y.V.
      • Rivas F.
      • Jinek M.
      • Wohlschlegel J.
      • Doudna J.A.
      • Chen C.Y.
      • Shyu A.B.
      • Yates 3rd, J.R.
      • Hannon G.J.
      • Filipowicz W.
      • Duchaine T.F.
      • Sonenberg N.
      Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.
      ). miRISC might further accelerate mRNA decay by recruiting decapping factors in a manner that is independent of their effects on deadenylation (
      • Nishihara T.
      • Zekri L.
      • Braun J.E.
      • Izaurralde E.
      miRISC recruits decapping factors to miRNA targets to enhance their degradation.
      ). In addition, the CCR4-NOT complex may play a role in miRNA-mediated translation repression (
      • Chekulaeva M.
      • Mathys H.
      • Zipprich J.T.
      • Attig J.
      • Colic M.
      • Parker R.
      • Filipowicz W.
      miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.
      ,
      • Braun J.E.
      • Huntzinger E.
      • Fauser M.
      • Izaurralde E.
      GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets.
      ). miRNA can induce translation repression independent of deadenylation (
      • Fukaya T.
      • Tomari Y.
      MicroRNAs mediate gene silencing via multiple different pathways in drosophila.
      ,
      • Mishima Y.
      • Fukao A.
      • Kishimoto T.
      • Sakamoto H.
      • Fujiwara T.
      • Inoue K.
      Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish.
      • Fukaya T.
      • Tomari Y.
      PABP is not essential for microRNA-mediated translational repression and deadenylation in vitro.
      ), and the CCR4-NOT complex does so in tethering experiments (
      • Chekulaeva M.
      • Mathys H.
      • Zipprich J.T.
      • Attig J.
      • Colic M.
      • Parker R.
      • Filipowicz W.
      miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.
      ,
      • Bawankar P.
      • Loh B.
      • Wohlbold L.
      • Schmidt S.
      • Izaurralde E.
      NOT10 and C2orf29/NOT11 form a conserved module of the CCR4-NOT complex that docks onto the NOT1 N-terminal domain.
      ). The interaction of a MIF4G domain of human CNOT1 with DDX6, through a structural arrangement that is analogous to the MIF4G domain of eIF4G and eIF4AI, contributes to the miRNA-mediated silencing (
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      ,
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ,
      • Rouya C.
      • Siddiqui N.
      • Morita M.
      • Duchaine T.F.
      • Fabian M.R.
      • Sonenberg N.
      Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1.
      ). These observations indicate that miRISC achieves post-transcriptional silencing via conserved but intricate functions of the CCR4-NOT complex.
      S. cerevisiae lacks the small RNA-producing enzyme Dicer and Ago and therefore does not produce canonical siRNAs and miRNAs. However, the basic machinery for controlling mRNA stability and translation, including the CCR4-NOT complex, decapping factors, and translation initiation factors, is highly conserved (
      • Parker R.
      RNA degradation in Saccharomyces cerevisae.
      ,
      • Coller J.
      • Parker R.
      Eukaryotic mRNA decapping.
      ). Notably, the yeast Pumilio-like protein Puf5/Mpt5 binds to the CCR4-NOT complex to silence and deadenylate specific mRNAs (
      • Goldstrohm A.C.
      • Seay D.J.
      • Hook B.A.
      • Wickens M.
      PUF protein-mediated deadenylation is catalyzed by Ccr4p.
      ,
      • Goldstrohm A.C.
      • Hook B.A.
      • Seay D.J.
      • Wickens M.
      PUF proteins bind Pop2p to regulate messenger RNAs.
      ), suggesting that the CCR4-NOT complex is involved in sequence-specific post-transcriptional regulation independent of the emergence of miRNAs. Previously, the Bartel and Roth laboratories (
      • Drinnenberg I.A.
      • Weinberg D.E.
      • Xie K.T.
      • Mower J.P.
      • Wolfe K.H.
      • Fink G.R.
      • Bartel D.P.
      RNAi in budding yeast.
      ,
      • Suk K.
      • Choi J.
      • Suzuki Y.
      • Ozturk S.B.
      • Mellor J.C.
      • Wong K.H.
      • MacKay J.L.
      • Gregory R.I.
      • Roth F.P.
      Reconstitution of human RNA interference in budding yeast.
      ) showed that gene silencing by siRNA could be reconstituted in S. cerevisiae by expressing either Saccharomyces castellii Ago1 and Dicer1 or human Ago2, Dicer, and TRBP.
      In this study, we successfully recapitulated two hallmarks of animal miRISC-mediated silencing in S. cerevisiae by tethering the middle domain of zebrafish TNRC6A to reporter mRNAs. Using mutant yeast strains, we showed that zebrafish TNRC6A directly stimulates decapping and 5′-to-3′ mRNA decay in a Not1-dependent but poly(A) tail- and translation-independent manner. In addition, we showed that the Dhh1 and Pat1 play crucial roles not only in stimulation of mRNA decapping but also in translational repression. These results indicate that the conserved architecture of Not1/CNOT1 provides a binding surface for TNRC6, thereby connecting miRISC to the decapping machinery and translation apparatus. Furthermore, miR-430-mediated mRNA decay was differentially susceptible to inhibition of deadenylation in zebrafish embryos. This tethering-based reconstitution system in yeast will complement miRNA studies in animal cells, in which genetic approaches are sometimes not applicable.

      DISCUSSION

      In this study, we established a heterologous experimental system by tethering animal TNRC6 proteins to mRNAs in yeast. Polysome analysis, pulse-labeling experiments, and measurement of mRNA half-lives revealed that the tethered Mid domain fragment of zebrafish TNRC6A induces mRNA degradation and translation repression in yeast. This result strongly suggests that miRISC induces both translation repression and mRNA degradation via interactions with fundamental factors that are conserved across a wide range of eukaryotes. Indeed, the highly conserved proteins Dhh1 and Pat1 mediate these two functions through the CCR4-NOT complex, which is recruited by the Mid domain (FIGURE 5., FIGURE 7.). These two factors are involved in miRNA-mediated silencing in animals (
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      ,
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ,
      • Behm-Ansmant I.
      • Rehwinkel J.
      • Doerks T.
      • Stark A.
      • Bork P.
      • Izaurralde E.
      mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
      ,
      • Eulalio A.
      • Rehwinkel J.
      • Stricker M.
      • Huntzinger E.
      • Yang S.F.
      • Doerks T.
      • Dorner S.
      • Bork P.
      • Boutros M.
      • Izaurralde E.
      Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing.
      ,
      • Fabian M.R.
      • Cieplak M.K.
      • Frank F.
      • Morita M.
      • Green J.
      • Srikumar T.
      • Nagar B.
      • Yamamoto T.
      • Raught B.
      • Duchaine T.F.
      • Sonenberg N.
      miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT.
      ,
      • Chekulaeva M.
      • Mathys H.
      • Zipprich J.T.
      • Attig J.
      • Colic M.
      • Parker R.
      • Filipowicz W.
      miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.
      • Braun J.E.
      • Huntzinger E.
      • Fauser M.
      • Izaurralde E.
      GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets.
      ,
      • Chu C.Y.
      • Rana T.M.
      Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.
      ), supporting the idea that TNRC6 can function in S. cerevisiae and suggesting that our system can be used to characterize the biologically relevant activities of miRISC. Although the data obtained in yeast with a truncated fragment of TNRC6 require careful validation in animal cells, the recapitulation of TNRC6-mediated silencing in yeast described here raises the possibility that genetic resources in yeast can be used to study basic principles of the miRNA system.
      The Mid domain of TNRC6A interacted with the yeast CCR4-NOT complex via CIM1 and W-motifs within the Mid domain that mediate the same interactions in animals (Fig. 1B) (
      • Fabian M.R.
      • Cieplak M.K.
      • Frank F.
      • Morita M.
      • Green J.
      • Srikumar T.
      • Nagar B.
      • Yamamoto T.
      • Raught B.
      • Duchaine T.F.
      • Sonenberg N.
      miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT.
      ,
      • Chekulaeva M.
      • Mathys H.
      • Zipprich J.T.
      • Attig J.
      • Colic M.
      • Parker R.
      • Filipowicz W.
      miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.
      ). This result, together with recent structural studies (
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      ,
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ), indicates that the conserved architecture of the CCR4-NOT complex provides a binding surface for GW182/TNRC6 proteins, thereby connecting miRISC to the mRNA fate modulators. Further biochemical and structural analysis in yeast will shed light on how this interaction evolved.
      The interactions of GW182/TNRC6 proteins with PABP and CCR4-NOT deadenylase play crucial roles in both translational repression and degradation of miRNA targets (
      • Fabian M.R.
      • Cieplak M.K.
      • Frank F.
      • Morita M.
      • Green J.
      • Srikumar T.
      • Nagar B.
      • Yamamoto T.
      • Raught B.
      • Duchaine T.F.
      • Sonenberg N.
      miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT.
      ,
      • Chekulaeva M.
      • Mathys H.
      • Zipprich J.T.
      • Attig J.
      • Colic M.
      • Parker R.
      • Filipowicz W.
      miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.
      • Braun J.E.
      • Huntzinger E.
      • Fauser M.
      • Izaurralde E.
      GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets.
      ,
      • Fabian M.R.
      • Mathonnet G.
      • Sundermeier T.
      • Mathys H.
      • Zipprich J.T.
      • Svitkin Y.V.
      • Rivas F.
      • Jinek M.
      • Wohlschlegel J.
      • Doudna J.A.
      • Chen C.Y.
      • Shyu A.B.
      • Yates 3rd, J.R.
      • Hannon G.J.
      • Filipowicz W.
      • Duchaine T.F.
      • Sonenberg N.
      Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.
      ). mRNA degradation by miRNAs and GW182/TNRC6 requires both deadenylase and the DCP1-DCP2 decapping complexes (
      • Behm-Ansmant I.
      • Rehwinkel J.
      • Doerks T.
      • Stark A.
      • Bork P.
      • Izaurralde E.
      mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
      ,
      • Chen C.Y.
      • Zheng D.
      • Xia Z.
      • Shyu A.B.
      Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.
      ). In addition, GW182 recruits decapping enzymes to target mRNAs independently of deadenylation (
      • Nishihara T.
      • Zekri L.
      • Braun J.E.
      • Izaurralde E.
      miRISC recruits decapping factors to miRNA targets to enhance their degradation.
      ). In this study, we demonstrated that the tethered TNRC6A fragment promoted decapping and 5′-to-3′ mRNA decay in a Not1-dependent but a poly(A) tail- and translation-independent manner (FIGURE 2., FIGURE 3.). The levels of decapped No-AUG-MS2-Rz mRNA were significantly increased in xrn1Δ cells expressing F-MS2-Mid (Fig. 3, B and C), indicating that the tethered Mid domain of TNRC6A recruits CCR4-NOT and the decapping complex and facilitates the decapping reaction independently of a poly(A) tail and translation. Together, these results suggest that mRNA decay caused by the Mid domain can mostly be attributed to the function of the yeast CCR4-NOT complex.
      Our results indicate that GW182/TNRC6 stimulates mRNA decay in yeast in a decapping-dependent but poly(A) tail- and translation-independent manner. We also found that tethering of the TNRC6A Mid domain degraded m7G-capped mRNA but not A-capped mRNA in zebrafish embryos (Fig. 4D). Moreover, miR-430 stimulated decapping and 5′-to-3′ degradation of eif4ebp2 mRNA in zebrafish embryos even when the deadenylase activity of the CCR4-NOT complex was not fully ensured (Fig. 4, F, G, and J). These results imply that, at least for some mRNAs like eif4ebp2, miRISC promotes mRNA decay from the 5′ end by directly stimulating decapping before (or in parallel to) deadenylation. Although more comprehensive study with multiple transcripts in diverse animals is prerequisite to generalize this degradation mode of miRNAs, it is noteworthy that some mRNAs that are degraded in an Ago1-dependent manner are degraded when CAF1 is knocked down in Drosophila S2 cells (
      • Behm-Ansmant I.
      • Rehwinkel J.
      • Doerks T.
      • Stark A.
      • Bork P.
      • Izaurralde E.
      mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
      ). Conversely, not all Ago1-RISC target mRNAs strictly require mRNA fate modulators for their decay (
      • Eulalio A.
      • Rehwinkel J.
      • Stricker M.
      • Huntzinger E.
      • Yang S.F.
      • Doerks T.
      • Dorner S.
      • Bork P.
      • Boutros M.
      • Izaurralde E.
      Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing.
      ). These observations suggest that each mRNA is differentially susceptible to decapping and deadenylation during the process of miRNA-mediated degradation.
      We found that rapid decay of No-AUG-MS2-Rz mRNA by the tethered TNRC6A Mid domain was abrogated in the dhh1Δpat1Δ double mutant but not in the dhh1Δ or pat1Δ single mutants (Fig. 5). We propose that Dhh1 and Pat1 contribute to decapping via distinct pathways in the absence of translation and a poly(A) tail. In addition to inhibiting translation, Dhh1 and Pat1 may have redundant functions in the formation of the decapping complex. Consequently, in the dhh1Δpat1Δ double mutant, the decapping complex may not be able to form, resulting in a very strong defect in decapping.
      A prevailing view, based on in vitro experiments, is that miRNAs inhibit translation at the initiation step by an as yet unknown mechanism (
      • Fukaya T.
      • Tomari Y.
      MicroRNAs mediate gene silencing via multiple different pathways in drosophila.
      ,
      • Zdanowicz A.
      • Thermann R.
      • Kowalska J.
      • Jemielity J.
      • Duncan K.
      • Preiss T.
      • Darzynkiewicz E.
      • Hentze M.W.
      Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression.
      ,
      • Thermann R.
      • Hentze M.W.
      Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation.
      • Mathonnet G.
      • Fabian M.R.
      • Svitkin Y.V.
      • Parsyan A.
      • Huck L.
      • Murata T.
      • Biffo S.
      • Merrick W.C.
      • Darzynkiewicz E.
      • Pillai R.S.
      • Filipowicz W.
      • Duchaine T.F.
      • Sonenberg N.
      MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F.
      ). A recent study has suggested that miRNAs repress translation initiation in a manner dependent upon eIF4AII, which interacts with CNOT7 of the CCR4-NOT complex in mammalian cells (
      • Meijer H.A.
      • Kong Y.W.
      • Lu W.T.
      • Wilczynska A.
      • Spriggs R.V.
      • Robinson S.W.
      • Godfrey J.D.
      • Willis A.E.
      • Bushell M.
      Translational repression and eIF4A2 activity are critical for microRNA-mediated gene regulation.
      ). However, because eIF4AII is present neither in Drosophila nor in unicellular eukaryotes, such as S. cerevisiae, it remains to be determined how this factor affects translation initiation in general. In this study, we showed that the tethered Mid domain of TNRC6A repressed translation in yeast (Fig. 1, G and H; see also Fig. 7B). Hence, the repressive activity of TNRC6A recapitulated in yeast is independent of eIF4AII. In addition, others also observed that eIF4AII does not interact directly with CNOT1 MIF4G domain (
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      ,
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ,
      • Rouya C.
      • Siddiqui N.
      • Morita M.
      • Duchaine T.F.
      • Fabian M.R.
      • Sonenberg N.
      Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1.
      ). Furthermore, recent in vitro studies showed that miRNAs trigger dissociation of eIF4A in Drosophila and both eIF4AI and eIF4AII in humans (
      • Fukao A.
      • Mishima Y.
      • Takizawa N.
      • Oka S.
      • Imataka H.
      • Pelletier J.
      • Sonenberg N.
      • Thoma C.
      • Fujiwara T.
      MicroRNAs trigger dissociation of eIF4AI and eIF4AII from target mRNAs in humans.
      ,
      • Fukaya T.
      • Iwakawa H.O.
      • Tomari Y.
      MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila.
      ). Apparently, more experiments will be necessary to determine the roles of eIF4AII in translation repression by miRNAs.
      In cells expressing F-MS2-Mid protein, the levels of GFP-MS2 or AUG-MS2 mRNAs were reduced in the polysome fraction, but increased in the 80 S monosome and free fractions, relative to cells expressing the control MS2 protein (Figs. 1G and 7B). This effect was observed in a reporter mRNA lacking a poly(A) tail (Figs. 1G and 7B). These results suggest that the tethered Mid domain of TNRC6A may block the formation of 48 S preinitiation complex or inhibit the steps after 80 S formation by CCR4-NOT complex independently of a poly(A) tail.
      How, then, does the CCR4-NOT complex inhibit translation? Polysome analysis and pulse-labeling experiments showed that translation repression by tethering of the Mid domain of TNRC6A was abrogated in dhh1Δ or pat1Δ single mutants as well as in the dhh1Δpat1Δ double mutant (Fig. 7). Translationally repressed mRNAs in wild type cells contained a cap structure (Fig. 3, D and E). Moreover, stimulation of mRNA decay from the 5′ end was impaired only in the dhh1Δpat1Δ double mutant, whereas translation repression by the tethered TNRC6A was suppressed in the dhh1Δ and pat1Δ single mutants (Fig. 7). These results clearly indicate that translation repression is not a consequence of decapping. The mRNA fate modulators Dhh1 and Pat1 independently repress translation in vivo, and they repress translation initiation in vitro by limiting the formation of a stable 48 S preinitiation complex (
      • Coller J.
      • Parker R.
      General translational repression by activators of mRNA decapping.
      ). Notably, a human homolog of Dhh1, DDX6/RCK1/p54, interacts with miRISC and contributes to translation repression in cultured cells (
      • Mathys H.
      • Basquin J.
      • Ozgur S.
      • Czarnocki-Cieciura M.
      • Bonneau F.
      • Aartse A.
      • Dziembowski A.
      • Nowotny M.
      • Conti E.
      • Filipowicz W.
      Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
      ,
      • Chen Y.
      • Boland A.
      • Kuzuoğlu-Öztürk D.
      • Bawankar P.
      • Loh B.
      • Chang C.T.
      • Weichenrieder O.
      • Izaurralde E.
      A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
      ,
      • Rouya C.
      • Siddiqui N.
      • Morita M.
      • Duchaine T.F.
      • Fabian M.R.
      • Sonenberg N.
      Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1.
      ,
      • Chu C.Y.
      • Rana T.M.
      Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.
      ), and GW182 associates with HPat in Drosophila cells (
      • Jäger E.
      • Dorner S.
      The decapping activator HPat a novel factor co-purifying with GW182 from Drosophila cells.
      ). Our results in yeast therefore support the model in which the CCR4-NOT complex mediates translation repression, at least in part, via recruitment of mRNA fate modulators through a direct interaction with Dhh1. Further experiments will be necessary to determine the conserved roles of decapping factors Dhh1/DDX6/RCK1/p54 and Pat1/HPat/PatL1 in translation repression by GW182/TNRC6 as well as other CCR4-NOT-interacting proteins.

      Acknowledgments

      We thank Dr. Yukihide Tomari for the kind gift of a reagent and for helpful discussions. We also thank members of our laboratories for discussion and critical comments on the manuscript.

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