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Inhibition of Nonsense-mediated mRNA Decay by the Natural Product Pateamine A through Eukaryotic Initiation Factor 4AIII*

Open AccessPublished:July 01, 2009DOI:https://doi.org/10.1074/jbc.M109.009985
      Nonsense-mediated mRNA decay (NMD) in mammalian cells is a key mechanism for the removal of mRNA containing premature stop codons and is mediated by the coordinated function of numerous proteins that dynamically associate with the exon junction complex. The information communicated by these interactions and the functional consequences from a mechanistic perspective, however, are not completely documented. Herein, we report that the natural product pateamine A (PatA) is capable of inhibiting NMD through direct interaction with eIF4AIII, which is independent of its inhibition of translation initiation. Furthermore, we have characterized the mechanisms by which PatA and cycloheximide modulate NMD. Unlike CHX, PatA was found to inhibit NMD by a novel mechanism that is independent of the phosphorylation of Up-frameshift protein 1.
      In mammalian cells, nonsense-mediated mRNA decay (NMD)
      The abbreviations used are: NMD
      nonsense-mediated mRNA decay
      PatA
      pateamine A
      DMDA-PatA
      desmethyl,desamino-pateamine A
      B-PatA
      biotinylated PatA
      TCR
      T-cell receptor
      HCV
      hepatitis C virus
      IRES
      internal ribosome entry site
      CrPV
      the cricket paralysis virus
      EJC
      the exon junction complex
      PTC
      premature stop codon
      CHX
      cycloheximide
      eIF
      eukaryotic initiation factor
      UPF
      up-frameshift
      CBP
      cap-binding protein.
      2The abbreviations used are: NMD
      nonsense-mediated mRNA decay
      PatA
      pateamine A
      DMDA-PatA
      desmethyl,desamino-pateamine A
      B-PatA
      biotinylated PatA
      TCR
      T-cell receptor
      HCV
      hepatitis C virus
      IRES
      internal ribosome entry site
      CrPV
      the cricket paralysis virus
      EJC
      the exon junction complex
      PTC
      premature stop codon
      CHX
      cycloheximide
      eIF
      eukaryotic initiation factor
      UPF
      up-frameshift
      CBP
      cap-binding protein.
      is one of the key RNA surveillance mechanisms to specifically degrade mRNA with premature stop codons (PTCs) located more than 50–55 nucleotides upstream of the final exon-exon junction. PTCs can be formed in genes containing a nonsense mutation or frameshift mutation or as a result of errors that occur during transcription or RNA splicing (
      • Chang Y.F.
      • Imam J.S.
      • Wilkinson M.F.
      ,
      • Weischenfeldt J.
      • Lykke-Andersen J.
      • Porse B.
      ,
      • Lejeune F.
      • Maquat L.E.
      ,
      • Singh G.
      • Lykke-Andersen J.
      ). After splicing, the exon junction complex (EJC) imprints mature mRNAs 20–24 nucleotides upstream of the exon-exon junction (
      • Le Hir H.
      • Izaurralde E.
      • Maquat L.E.
      • Moore M.J.
      ). The EJC is a dynamic multiprotein complex that plays an essential role in NMD. The core EJC proteins eIF4AIII, Y14, Magoh, and MLN51 form a platform to interact with several other proteins in a dynamic fashion to regulate NMD (
      • Stroupe M.E.
      • Tange T.Ø.
      • Thomas D.R.
      • Moore M.J.
      • Grigorieff N.
      ). The spatial-temporal regulation of NMD by the EJC and its partner proteins has been extensively investigated, leading to the proposition of the “linear interaction model” (
      • Singh G.
      • Lykke-Andersen J.
      ,
      • Chamieh H.
      • Ballut L.
      • Bonneau F.
      • Le Hir H.
      ). According to this model, deposition of the EJC onto mRNA causes the Y14-Magoh and eIF4AIII complex to effectively recruit Upf3 that interacts with Upf2. Although the mechanism of loading Upf1 onto EJC is poorly understood, it has been shown that phosphorylation and dephosphorylation of Upf1 by SMG-1, -5, -6, and -7 affect the interaction with the EJC complex through Upf2 or Upf3, causing dissociation from ribosome. Association of Upf1 with the EJC remodels the EJC to expose the mRNA for degradation (
      • Kashima I.
      • Yamashita A.
      • Izumi N.
      • Kataoka N.
      • Morishita R.
      • Hoshino S.
      • Ohno M.
      • Dreyfuss G.
      • Ohno S.
      ,
      • Page M.F.
      • Carr B.
      • Anders K.R.
      • Grimson A.
      • Anderson P.
      ,
      • Chiu S.Y.
      • Serin G.
      • Ohara O.
      • Maquat L.E.
      ,
      • Ohnishi T.
      • Yamashita A.
      • Kashima I.
      • Schell T.
      • Anders K.R.
      • Grimson A.
      • Hachiya T.
      • Hentze M.W.
      • Anderson P.
      • Ohno S.
      ,
      • Yamashita A.
      • Kashima I.
      • Ohno S.
      ,
      • Grimson A.
      • O'Connor S.
      • Newman C.L.
      • Anderson P.
      ). Most recently, SMG6 was identified as an endonuclease to cleave the aberrant mRNA near the PTC (
      • Eberle A.B.
      • Lykke-Andersen S.
      • Mühlemann O.
      • Jensen T.H.
      ).
      It has been demonstrated that NMD occurs in the cytoplasm and is coupled with translation to inspect mRNA for PTCs through ribosome scanning (
      • Singh G.
      • Jakob S.
      • Kleedehn M.G.
      • Lykke-Andersen J.
      ,
      • Clement S.L.
      • Lykke-Andersen J.
      ,
      • Carter M.S.
      • Doskow J.
      • Morris P.
      • Li S.
      • Nhim R.P.
      • Sandstedt S.
      • Wilkinson M.F.
      ,
      • Wang J.
      • Vock V.M.
      • Li S.
      • Olivas O.R.
      • Wilkinson M.F.
      ,
      • Wilkinson M.F.
      ). The fact that all known translation inhibitors were found to block NMD supports this model (
      • Carter M.S.
      • Doskow J.
      • Morris P.
      • Li S.
      • Nhim R.P.
      • Sandstedt S.
      • Wilkinson M.F.
      ,
      • Noensie E.N.
      • Dietz H.C.
      ). Some studies have suggested that ribosome scanning that displaces EJCs occurs during the “pioneer round” of translation mediated by the association of nuclear cap-binding proteins (CBPs) such as CBP20 and CBP80 with NMD-targeted mRNAs as opposed to the subsequent “steady-state” translation via eukaryotic translation initiation factor 4A (
      • Ishigaki Y.
      • Li X.
      • Serin G.
      • Maquat L.E.
      ). However, another study suggested that NMD can occur independently of the CBPs using encephalomyocarditis virus and hepatitis C virus (HCV) internal ribosome entry site (IRES)-initiated mRNA, which is translated independently of the cap (
      • Holbrook J.A.
      • Neu-Yilik G.
      • Gehring N.H.
      • Kulozik A.E.
      • Hentze M.W.
      ,
      • Isken O.
      • Kim Y.K.
      • Hosoda N.
      • Mayeur G.L.
      • Hershey J.W.
      • Maquat L.E.
      ).
      Pateamine A (PatA) was first isolated from the marine sponge Mycale sp. and shown to possess potent anti-proliferative and immunosuppressive activities (
      • Northcote P.T.
      • Blunt J.W.
      • Munro M.H.
      ,
      • Hood K.A.
      • West L.M.
      • Northcote P.T.
      • Berridge M.V.
      • Miller J.H.
      ,
      • Romo D.
      • Choi N.S.
      • Li S.
      • Buchler I.
      • Shi Z.
      • Liu J.O.
      ,
      • Romo D.
      • Rzasa R.M.
      • Shea H.A.
      • Park K.
      • Langenhan J.M.
      • Sun L.
      • Akhiezer A.
      • Liu J.O.
      ,
      • West L.M.
      • Northcote P.T.
      • Battershill C.N.
      ). Recently, we and others demonstrated that PatA specifically binds to eukaryotic translation initiation factor 4A I/II to stabilize its closed conformation, leading to the inhibition of eukaryotic translation initiation by perturbing the eIF4F complex, which is composed of translation initiation factors eIF4E, eIF4G, and eIF4AI/II (
      • Dang Y.
      • Kedersha N.
      • Low W.K.
      • Romo D.
      • Gorospe M.
      • Kaufman R.
      • Anderson P.
      • Liu J.O.
      ,
      • Low W.K.
      • Dang Y.
      • Bhat S.
      • Romo D.
      • Liu J.O.
      ,
      • Low W.K.
      • Dang Y.
      • Schneider-Poetsch T.
      • Shi Z.
      • Choi N.S.
      • Merrick W.C.
      • Romo D.
      • Liu J.O.
      ,
      • Bordeleau M.E.
      • Matthews J.
      • Wojnar J.M.
      • Lindqvist L.
      • Novac O.
      • Jankowsky E.
      • Sonenberg N.
      • Northcote P.
      • Teesdale-Spittle P.
      • Pelletier J.
      ). The eIF4A proteins are the prototypical D-E-A-D box proteins, having RNA-dependent ATPase activity and ATP-dependent RNA helicase activity (
      • Linder P.
      ). In addition to eIF4AI/II, PatA was also shown to bind the closely related homolog eIF4AIII and stimulate its ATPase activity (
      • Low W.K.
      • Dang Y.
      • Bhat S.
      • Romo D.
      • Liu J.O.
      ,
      • Bordeleau M.E.
      • Matthews J.
      • Wojnar J.M.
      • Lindqvist L.
      • Novac O.
      • Jankowsky E.
      • Sonenberg N.
      • Northcote P.
      • Teesdale-Spittle P.
      • Pelletier J.
      ). The main cellular function of eIF4AIII is to serve as a core component of the EJC (
      • Shibuya T.
      • Tange T.Ø.
      • Sonenberg N.
      • Moore M.J.
      ,
      • Ferraiuolo M.A.
      • Lee C.S.
      • Ler L.W.
      • Hsu J.L.
      • Costa-Mattioli M.
      • Luo M.J.
      • Reed R.
      • Sonenberg N.
      ,
      • Ballut L.
      • Marchadier B.
      • Baguet A.
      • Tomasetto C.
      • Séraphin B.
      • Le Hir H.
      ,
      • Palacios I.M.
      • Gatfield D.
      • St Johnston D.
      • Izaurralde E.
      ,
      • Shibuya T.
      • Tange T.Ø.
      • Stroupe M.E.
      • Moore M.J.
      ).
      Given the known cellular function of eIF4AIII in NMD, we have sought to distinguish the two effects of PatA as mediated through perturbation of its different targets, eIF4AI/II and eIF4AIII, and to determine if PatA has an impact on NMD through its interaction with eIF4AIII in addition to its indirect effect on NMD via inhibition of eIF4AI/II-mediated translation initiation. Using a well studied T-cell receptor β (TCR-β) reporter system, we observed inhibition of NMD by PatA and its active analog desmethyl,desamino-PatA (DMDA-PatA) (
      • Romo D.
      • Choi N.S.
      • Li S.
      • Buchler I.
      • Shi Z.
      • Liu J.O.
      ,
      • Low W.K.
      • Dang Y.
      • Schneider-Poetsch T.
      • Shi Z.
      • Choi N.S.
      • Merrick W.C.
      • Romo D.
      • Liu J.O.
      ) through specific interaction with eIF4AIII. We have demonstrated that this eIF4AIII-dependent inhibition of NMD by PatA is likely mediated through stabilization of the interaction between Upf1 and the ECJ complex. Furthermore, we found that PatA affected the dynamic association of Upf1 to the EJC without affecting Upf1 phosphorylation. This is in contrast to cycloheximide (CHX), a translation elongation inhibitor that also inhibits NMD. Together, these results shed new light on the regulation of NMD by the EJC and Upf proteins and suggest that PatA may serve as a new molecular probe of the NMD process.

      Discussion

      PatA inhibits eukaryotic translation initiation through binding to eIF4AI (
      • Low W.K.
      • Dang Y.
      • Schneider-Poetsch T.
      • Shi Z.
      • Choi N.S.
      • Merrick W.C.
      • Romo D.
      • Liu J.O.
      ,
      • Bordeleau M.E.
      • Matthews J.
      • Wojnar J.M.
      • Lindqvist L.
      • Novac O.
      • Jankowsky E.
      • Sonenberg N.
      • Northcote P.
      • Teesdale-Spittle P.
      • Pelletier J.
      ) and likely disrupting the eIF4F complex (
      • Low W.K.
      • Dang Y.
      • Schneider-Poetsch T.
      • Shi Z.
      • Choi N.S.
      • Merrick W.C.
      • Romo D.
      • Liu J.O.
      ). Herein, we have demonstrated that PatA is also capable of inhibiting NMD of mRNA through directly targeting eIF4AIII, a core component of the EJC. That is, for a specific mRNA such as the HCV or CrPV IRES-driven reporters where translation is refractory to PatA, NMD can still be inhibited by PatA. This finding is consistent with the previous observation that knockdown of both eIF4AI and eIF4AII had no effect on the NMD pathway (
      • Ferraiuolo M.A.
      • Lee C.S.
      • Ler L.W.
      • Hsu J.L.
      • Costa-Mattioli M.
      • Luo M.J.
      • Reed R.
      • Sonenberg N.
      ). Although it is formally possible that inhibition of NMD is secondary to the induction of stress granules by PatA followed by the movement of PTC-containing mRNA to stress granules, this is unlikely as the two IRES-driven reporter mRNAs were insensitive to PatA for translation while the corresponding NMD were inhibited.
      Although translation elongation inhibitors such as CHX and puromycin have been reported to inhibit NMD (
      • Carter M.S.
      • Doskow J.
      • Morris P.
      • Li S.
      • Nhim R.P.
      • Sandstedt S.
      • Wilkinson M.F.
      ,
      • Noensie E.N.
      • Dietz H.C.
      ), we demonstrated that PatA, a translation initiation inhibitor, is also capable of inhibiting NMD. It is thus not surprising that the molecular mechanisms of inhibition of NMD by PatA and CHX differ from each other. We observed that in the presence of DMDA-PatA, co-immunoprecipitation of PTC-containing mRNA with eIF4AIII or Y14 was increased compared with control, whereas no change or slight change was observed for the eIF4AIII or Y14-bound PTC-containing mRNA in the presence of CHX (Fig. 4). Furthermore, CHX induced phosphorylation of Upf1, whereas phosphorylation levels were unchanged, or slightly decreased under DMDA-PatA treatment (Fig. 5B). Together, these observations suggest the distinct mechanisms of inhibition of NMD by the two compounds. We surmise that these differences are a consequence of the direct action of PatA on EJC through its binding to eIF4AIII.
      Activation of NMD has been proposed to occur by a series of protein-protein interactions. First, the presence of PTCs prevents the removal of EJCs from the mRNA, leading to recruitment of Upf3 through its interaction with the EJC core, which in turn recruits Upf2. Upf2 and Upf3a/b mediate the recruitment of Upf1 to EJC through their concurrent interactions with Upf1 and the binding site formed by Y14-Magoh and eIF4AIII (
      • Chamieh H.
      • Ballut L.
      • Bonneau F.
      • Le Hir H.
      ), respectively, and their mutual interaction (
      • Chamieh H.
      • Ballut L.
      • Bonneau F.
      • Le Hir H.
      ,
      • Gehring N.H.
      • Neu-Yilik G.
      • Schell T.
      • Hentze M.W.
      • Kulozik A.E.
      ,
      • Le Hir H.
      • Gatfield D.
      • Izaurralde E.
      • Moore M.J.
      ,
      • Lykke-Andersen J.
      ). In addition, after the ribosome is stalled at the PTC, Upf1 can form SURF complex with eRF3, eRF1, and SMG-1. The dynamic association of Upf1 with two complexes plays an essential role in NMD (
      • Chang Y.F.
      • Imam J.S.
      • Wilkinson M.F.
      ,
      • Maquat L.E.
      ,
      • Isken O.
      • Maquat L.E.
      ), thus signaling the dissociation of ribosome from mRNA and the eventual degradation of the PTC-containing mRNA (
      • Chang Y.F.
      • Imam J.S.
      • Wilkinson M.F.
      ). In two independent studies, CHX has been shown to interfere with NMD and to cause phosphorylation of Upf1, respectively. In this study, we found that CHX induced phosphorylation of Upf1 led to its stable association with Upf3b and the associated EJC·mRNA complex. This is consistent with the observations made by two independent groups that the phosphorylated Upf1 significantly enhances the interaction with other components of NMD pathway (
      • Isken O.
      • Kim Y.K.
      • Hosoda N.
      • Mayeur G.L.
      • Hershey J.W.
      • Maquat L.E.
      ,
      • Ivanov P.V.
      • Gehring N.H.
      • Kunz J.B.
      • Hentze M.W.
      • Kulozik A.E.
      ). Based on our experimental evidence, binding of PatA to eIF4AIII initiates a cascade of conformational changes by trapping eIF4AIII in an otherwise unstable conformation. This conformational change is propagated through other proteins of the EJC complex to Upf3 and subsequently Upf2 or Upf1. Thus, PatA and CHX act at different “ends” of the NMD process but achieve the same outcome: stabilization of the EJC·Upf1 interaction, perturbation of the dynamic interaction of Upf1 with the EJC complex, and the inhibition of NMD.
      We have previously shown that PatA may act by “trapping” eIF4AI in a “closed” conformation, which leads to its increased enzymatic activity and prevents the necessary cycling through the different conformational states associated with its normal function (
      • Low W.K.
      • Dang Y.
      • Bhat S.
      • Romo D.
      • Liu J.O.
      ). The increased association of RNA with the EJC in the presence of DMDA-PatA and the inhibition of NMD observed here suggest that PatA may exert similar conformational effects on eIF4AIII. The presence of PatA may trap eIF4AIII in the closed conformation as observed in structural studies of the EJC (
      • Andersen C.B.
      • Ballut L.
      • Johansen J.S.
      • Chamieh H.
      • Nielsen K.H.
      • Oliveira C.L.
      • Pedersen J.S.
      • Séraphin B.
      • Le Hir H.
      • Andersen G.R.
      ,
      • Bono F.
      • Ebert J.
      • Lorentzen E.
      • Conti E.
      ) and does not permit its cycling through different conformations. Furthermore, this mode of action of PatA on eIF4AIII would again be consistent with proposed models for DDX proteins where a tightly regulated cycle of conformational changes and ATP hydrolysis are necessary for correct ribonucleoprotein particle remodeling (
      • Linder P.
      ). For both eIF4AI and eIF4AIII, PatA stimulates ATPase activity in addition to increasing RNA affinity. In fact, ATP stimulation of RNA binding to eIF4AI was no longer required in the presence of PatA (
      • Bordeleau M.E.
      • Matthews J.
      • Wojnar J.M.
      • Lindqvist L.
      • Novac O.
      • Jankowsky E.
      • Sonenberg N.
      • Northcote P.
      • Teesdale-Spittle P.
      • Pelletier J.
      ), and we have proposed that PatA may decouple RNA release by eIF4AI during ATP hydrolysis (
      • Low W.K.
      • Dang Y.
      • Bhat S.
      • Romo D.
      • Liu J.O.
      ). Loss of ATPase mutations in eIF4AIII have been shown to be inconsequential to NMD, whereas loss of RNA-binding mutations are necessary to disrupt NMD (
      • Shibuya T.
      • Tange T.Ø.
      • Stroupe M.E.
      • Moore M.J.
      ). Thus, the increased affinity of the EJC for RNA may be due to DMDA-PatA trapping eIF4AIII in a high RNA affinity conformation and not allowing for proper cycling of conformations needed for dynamic remodeling of the EJC. In addition, Magoh-Y14 inhibits the ATPase activity of eIF4AIII, which would increase eIF4AIII affinity for RNA, thus anchoring eIF4AIII onto RNA (
      • Ballut L.
      • Marchadier B.
      • Baguet A.
      • Tomasetto C.
      • Séraphin B.
      • Le Hir H.
      ). Accordingly, under “normal” conditions, eIF4AIII binding to RNA is dependent on the integrity of the EJC core. However, in the presence of PatA, eIF4AIII is anchored onto RNA independent of ATP hydrolysis and trapped in a distinct conformation from its drug-free form. Given the extensive contacts between eIF4AIII and other components of the EJC complex, the alteration in the conformation of eIF4AIII inevitably causes conformational changes of other proteins within the EJC·Upf complex.
      The interaction of the EJC with mRNA is a crucial marker for targeting aberrant mRNA for NMD. We observed higher association of PTC-containing mRNA with eIF4AIII or Y14 under DMDA-PatA treatment compared with control (Fig. 4). Furthermore, co-immunoprecipitation analysis of EJC proteins demonstrated no changes in Magoh and Y14 when eIF4AIII was immunoprecipitated. Based on these observations, we conclude that the EJC is more tightly associated with the mRNA as it has been demonstrated that Y14 does not bind RNA on its own, and is associated with RNA through its binding to eIF4AIII (
      • Ballut L.
      • Marchadier B.
      • Baguet A.
      • Tomasetto C.
      • Séraphin B.
      • Le Hir H.
      ). These results also support the notion that PatA directly acts on eIF4AIII and the enhanced association with PTC-containing RNA in the presence of DMDA-PatA is likely a consequence of the conformational change of eIF4AIII similar to eIF4AI (
      • Low W.K.
      • Dang Y.
      • Bhat S.
      • Romo D.
      • Liu J.O.
      ). For CHX, the slight differences from DMSO control were observed for PTC-containing mRNA, in contrast to WT mRNA, where both DMDA-PatA and CHX treatments caused increased association of the EJC with mRNA. Our co-immunoprecipitation results also demonstrated that the interactions of the core EJC proteins were not changed from carrier control for CHX, suggesting that, in the presence of CHX, the EJC is not tightly associated with PTC-containing mRNA.
      The increase in EJC association with WT reporter mRNA in the presence of CHX (Fig. 4) may be a consequence of translation inhibition where EJCs are not removed by the ribosome, whereas for DMDA-PatA the direct action of trapping eIF4AIII caused the similar end result. For CrPVPTC, unlike the situation for WT, the presence of the PTC induces NMD. Thus, lack of accumulation of EJCs under CHX treatment may indicate a normal NMD function of EJC removal is not inhibited, whereas as the global NMD process is inhibited, i.e. CHX inhibits a step following EJC removal. For DMDA-PatA, direct action on eIF4AIII results in increased EJC association with the RNA regardless of the presence of a PTC.
      Given the multitude of proteins involved in the formation of the EJC·Upf ternary complex during NMD, we extensively examined the effects of DMDA-PatA on the association of Upf1 with the remaining complex by its co-immunoprecipitation with a number of proteins. Although an increase in interaction was seen with Upf2, eIF4AIII, and Y14 in the presence of CHX or DMDA-PatA when Upf1 was immunoprecipitated, no reciprocal increase in Upf1 was observed when Upf2 was pulled down, suggesting that Upf2 may not be a relevant mediator of the effect of DMDA-PatA on NMD. In contrast, consistent enhancement was seen for the interaction between Upf3b and Upf1 in reciprocal co-immunoprecipitation experiments. It is thus highly likely that the increase in the interaction between Upf1 and EJC is mediated by Upf3b independent of Upf2. This is also consistent with recent evidence that Upf1 is capable of independently interacting with Upf2 and Upf3b (
      • Ivanov P.V.
      • Gehring N.H.
      • Kunz J.B.
      • Hentze M.W.
      • Kulozik A.E.
      ). Although a mutant of Upf1 (VV204–205DI) is functional in NMD and can be phosphorylated, it only interacts with Upf3b, but not Upf2 (
      • Ivanov P.V.
      • Gehring N.H.
      • Kunz J.B.
      • Hentze M.W.
      • Kulozik A.E.
      ). Thus, the inhibitory effects of PatA and CHX on NMD are mediated by Upf3b to cause an increase in Upf1 associated with EJC and its phosphorylation in the case of CHX.
      It has been reported that CHX can change the phosphorylation status of Upf1, which is also sensitive to phosphatidylinositol 3-kinase inhibitors (
      • Pal M.
      • Ishigaki Y.
      • Nagy E.
      • Maquat L.E.
      ). However, the mechanism of up-regulation of Upf1 phosphorylation by CHX has remained unclear. Does CHX directly activate the kinase of Upf1, or is Upf1 phosphorylation indirectly caused by stabilization of the polysome? This question remains to be answered and is beyond the scope of this study. However, an important observation from our studies was that PatA enhanced the interaction of Upf1 with the EJC independent of Upf1 phosphorylation. This is consistent with our model in which the increase in Upf1 interaction with the EJC complex is mediated through conformational changes.

      Acknowledgments

      We are grateful to Drs. Paul Englund, Jerry Hart, and Daniel Lane for generous provision of advice and access to special equipments. We thank Dr. Joshua Mendell, Dr. Peter Sarnow, Dr. Jens Lykke-Andersen, Dr. Jerry Pelletier, and Sara Cooke for plasmids, antibodies, and technique supports. We thank Benjamin Nacev and David Walker for assistance with preparation of the manuscript.

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