Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells

    Insulin signaling augments eIF4E-dependent nonsense-mediated mRNA decay in mammalian cells Jungyun Park, Seyoung Ahn, Aravinth K. Jayabalan, Takbum Ohn, Hyun Chul Koh, Jungwook Hwang PII: DOI: Reference:

S1874-9399(15)00270-9 doi: 10.1016/j.bbagrm.2015.12.006 BBAGRM 975

To appear in:

BBA - Gene Regulatory Mechanisms

Received date: Revised date: Accepted date:

16 July 2015 14 December 2015 17 December 2015

Please cite this article as: Jungyun Park, Seyoung Ahn, Aravinth K. Jayabalan, Takbum Ohn, Hyun Chul Koh, Jungwook Hwang, Insulin signaling augments eIF4E-dependent nonsense-mediated mRNA decay in mammalian cells, BBA - Gene Regulatory Mechanisms (2015), doi: 10.1016/j.bbagrm.2015.12.006

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ACCEPTED MANUSCRIPT Manuscript No.: BBAGRM-15-155R1 Insulin signaling augments eIF4E-dependent nonsense-mediated mRNA decay in mammalian

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cells

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Jungyun Park a, Seyoung Ahna, Aravinth K. Jayabalanc, Takbum Ohnc, Hyun Chul Kohd,*, and Jungwook Hwanga,b*

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Graduate School for Biomedical Science & Engineering, Hanyang University, Seoul, Korea; Department of Medical Genetics, College of Medicine, Hanyang University, Seoul; cDepartment of

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Cellular and Molecular Medicine, College of Medicine, Chosun University, Gwangju, Korea; Department of Pharmacology, College of Medicine, Hanyang University, Seoul, Korea

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Jungwook Hwang

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*To whom correspondence should be addressed:

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Graduate School for Biomedical Science & Engineering and Department of Medical Genetics, College of Medicine, FTC1202-8, Hanyang University, 222 Wangimni-ro, Seongdong-gu, Seoul 04763, Korea

E-mail: [email protected] Tel: +82-2-2220-2427 Fax: +82-2-2220-2422

Hyun Chul Koh Department of Pharmacology, College of Medicine, Hanyang University, 222 Wangimni-ro, Seongdong-gu, Seoul 04763, Korea

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ACCEPTED MANUSCRIPT E-mail: [email protected] Tel: +82-2-2220-0653

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Running title: Enhancement of translation augments NMD

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Fax: +82-2-2292-6686

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ACCEPTED MANUSCRIPT ABSTRACT Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature

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termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to

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impair NMD; however, few studies have investigated the correlation between enhanced translation

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and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an

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increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation

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increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing

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bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound

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NMD targets.

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Key words: PI3K/AKT/mTOR, insulin, NMD, translation, processing body

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ACCEPTED MANUSCRIPT 1. Introduction In mammalian cells, nonsense-mediated mRNA decay (NMD) is a key pathway through which the

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abundance of newly synthesized transcripts is regulated. In this pathway, premature termination

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codon (PTC)-harboring transcripts and natural transcripts containing a long 3’ untranslated region

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(UTR), a 5’ upstream open reading frame (uORF), or an intron in a 3’UTR are eliminated in a translation-dependent manner ([1-3] and references therein). Spliced transcripts with a 7methylguanylate cap at the 5’ end are bound by one of the cap-binding proteins (CBP)20-CBP80 or

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eukaryotic translation-initiation factor 4E (eIF4E), and it is these complexes that serve as templates

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for translation. Translation is initiated by binding of the CTIF and eIF4G scaffold proteins to capbinding proteins CBP20/80 or eIF4E respectively, recruiting ribosomal subunits and translation

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initiation factors to the mRNA and initiating the NMD pathway [4-9]. Interestingly, two recent

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studies revealed that the interaction of eIF4G with poly(A) binding protein (PABPC1) suppresses exon-junction complex (EJC)-independent NMD, providing the mechanism through which long

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3’UTR-containing transcripts and translation initiation proteins are involved in NMD, although the decay of mRNA containing long 3’UTRs can also be caused by cis-element sequences [10-12].

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Translation plays a pivotal role in canonical EJC-dependent NMD. This is supported by evidence showing that EJC-dependent NMD requires the SURF complex, which consists of suppressor with a morphogenic effect on genitalia 1 (SMG1) protein, UPF1, eukaryotic release factor 1 (eRF1), and eRF3 and is formed immediately after a scanning ribosome recognizes a PTC [13]. After formation, the SMG1-UPF1 complex localizes to EJC-bound UPF2 with the help of DHX34, resulting in UPF1 phosphorylation and the recruitment of various decay factors including SMG5/7, SMG6, and PNRC2 [13-19]. The ATP-dependent RNA helicase UPF1 interacts both directly and indirectly with various NMD factors [20]. Interestingly, UPF1 resides on transcripts regardless of the occurrence of NMD, indicating that binding of UPF1 to transcripts does not specify NMD targets. For example, the long

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ACCEPTED MANUSCRIPT noncoding RNA MALAT1, which is restricted to the nucleus and is not translated, coimmunoprecipitates with UPF1. This finding suggests that UPF1 immediately associates with

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nascent transcripts [21]. During ribosome scanning, UPF1 is removed from the coding sequence of

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transcripts [22]; however, bound UPF1 remains on the 3’UTR since ribosomes do not move past the

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stop codon. This finding explains why UPF1 is enriched in PTC-containing transcripts compared with PTC-free transcripts, since PTC-containing transcripts have longer 3’UTRs. In support of this, Zünd et al. [21] reported that puromycin treatment shifts UPF1 binding from the 3’UTR to coding

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regions [23]. Moreover, inhibition of translation by using translation elongation inhibitors has been

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shown to impair NMD, reducing the amount of NMD target transcripts that coimmunoprecipitate with UPF1 [24]. These data suggest that translation elongation is important for UPF1 association

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with transcripts.

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Translation enhancement can be triggered by insulin treatment via PI3K/AKT/mTOR signaling [25, 26]. Insulin activates phosphoinositide 3-kinase (PI3K), whose actions are opposed by

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those of phosphatase and tensin homologue (PTEN), which hydrolyzes the 3-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PIP3). Loss of PTEN augments the activity of AKT (also

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known as protein kinase B [PKB]), leading to enhanced cell cycle progression, cell growth, and cellular metabolism [27, 28]. These effects are mediated by the downregulation of phosphorylated glycogen synthase kinase 3 (GSK3), forkhead box O (FOXO), and p27 [29]. Activated AKT is also able to activate mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates eIF4E-binding protein (4EBP1) thereby activating protein translation and cell survival [27]. Here we report that increased translation induced by insulin-mediated PI3K/AKT/mTOR signaling augments NMD. Rapamycin treatment in the presence of insulin stimulation impaired this increase in NMD, whereas increasing the levels of phosphorylated AKT and 4EBP with a PTEN inhibitor augmented NMD. Furthermore, downregulation of AKT resulted in upregulation of both exogenous and endogenous NMD targets. Interestingly, insulin stimulation augmented UPF1 binding

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ACCEPTED MANUSCRIPT to both PTC-containing transcripts and eIF4E-bound transcripts, and upregulation of NMD by insulin stimulation was inhibited by the expression of an unphosphorylated 4EBP1 dominant-

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negative mutant. Furthermore, the colocalization of DCP1a with UPF1 or eIF4E was enhanced by

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insulin stimulation. Taken together, our data indicate that upregulation of translation by insulin

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stimulation enhances the degradation of PTC-containing transcripts (presumably eIF4E-bound) via

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PI3K/AKT/mTOR signaling.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Cell culture, transfection, and drug treatments

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HeLa cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with

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10% fetal bovine serum and 1% penicillin/streptomycin. When specified, cells were transiently

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transfected with plasmid DNA or siRNA using Lipofectamine 2000 (Invitrogen) or Oligofectamine (Invitrogen), respectively. One day after siRNA transfection, cells were transfected with NMD test plasmids [pmCMV-Gl (β-globin) or pmCMV-GPx1 (glutathione peroxidase 1), either PTC-free

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(Norm) or PTC-containing (Ter)] and a reference plasmid [phCMV-MUP (mouse major urinary

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protein)] (Fig. 1A). When specified, cells were transfected with NMD test plasmids that included pmCMV-Gl Norm or Ter lacking intron 2 [30] or RLuc-Gl [24] with pGL3 (Promega) or pCI-neoFLAG-UPF1 [6]. Unless otherwise specified, serum depletion and insulin stimulation were carried

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out by briefly washing cells with DMEM followed by incubation with serum-free DMEM for 24 h

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and stimulation with insulin (1 μM) for 4 h. When noted, cells were treated with 20 nM PTEN-I,

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(VO-OHpic trihydrate, a PTEN inhibitor; Sigma) for 24 h, 10 μM wortmannin for 4 h, 1 μM insulin for 4 h, or 100 nM rapamycin for 4 h. Transcription was inhibited with 5,6-dichloro-1-β-D-

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ribofuranosylbenzimidazole (DRB) during insulin stimulation. Cells were lysed in a hypotonic solution as previously described [31].

2.2. siRNA-mediated gene silencing To silence the expression of specific genes, HeLa cells were seeded one day before transfection. Cells were transiently transfected with the siRNAs listed in Supplementary Table S1 or nonspecific control siRNA (Bioneer, Korea) using Oligofectamine and harvested three days later.

2.3. Immunoprecipitation

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ACCEPTED MANUSCRIPT Cell lysates were generated from HeLa cells that had been transiently transfected with NMD test plasmids and reference plasmids or with pCI-Neo-FLAG-UPF1 (FLAG-tagged UPF1) and, as

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specified, co-transfected with HA-tagged 4EBP1-TTAA. Immunoprecipitation (IP) was performed

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with anti-UPF1 antibodies, FLAG antibody-conjugated beads (Sigma), or anti-CBP80 antibodies as

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previously described [31, 32]. Coimmunoprecipitated proteins and mRNA were analyzed by Western blotting and quantitative RT-PCR (RT-qPCR), respectively.

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2.4. Western blotting

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Proteins present in cell lysates and IP eluates were separated by electrophoresis on 7-15% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (Millipore). Blots were

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probed with antibodies against the following proteins: UPF1 (a gift from Dr. Lynne E. Maquat);

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phosphorylated UPF1 (a gift from Dr. Yoon Ki Kim [16]); SMG1, AKT, phosphorylated AKT, 4EBP1, phosphorylated 4EBP1, mTOR, phosphorylated mTOR, PTEN, GSK3β, phosphorylated

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GSK3β, p70S6K, phosphorylated p70S6K, RPS3 (all from Cell Signaling Technology); IRS1, calnexin, β-actin, PLCγ, p62, PABP, UPF2, eIF3b (all from Santa Cruz Biotechnology); CBP80

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(Cell Signaling for Western blotting and IP; a gift from Dr. Yoon Ki Kim for immunofluorescence analysis [33]); eIF4E (Cell Signaling Technology for Western blotting; Santa Cruz Biotechnology for immunofluorescence analysis); HA (Roche); DCP1a (Bethyl Laboratories), and FLAG (Genscript).

2.5. Quantitative and semiquantitative RT-PCR Quantitative RT-PCR (RT-qPCR) was performed as previously described [31] using the primers listed in Supplementary Table S1. The primers used to amplify GAPDH and MUP in sqPCR were the same as those used for RT-qPCR.

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ACCEPTED MANUSCRIPT 2.6. Cell fractionation Nuclear and cytoplasmic HeLa fractions were prepared using Nuclear and Cytoplasmic Extraction

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Reagents (Thermo Scientific) according to the manufacturer’s protocol.

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2.7. Immunofluorescence

To determine whether DCP1a colocalized with UPF1, eIF4E, or CBP80, HeLa cells were transiently transfected with mCherry-DCP1a, incubated in serum-depleted DMEM medium for 24 h, and then

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stimulated with insulin. Fixation and staining procedures have been previously described [31].

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Immunofluorescence images were obtained using a confocal microscope (Leica, TCS SP5) at the factory settings for the appropriate excitation and emission wavelengths of Alexa Fluor 488. Nuclei

2.8. Polysome profiling

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were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Vector).

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HeLa cells were transiently transfected with NMD test and reference plasmids. Cells were serumstarved for 24 h post-transfection and then incubated in the absence or presence of 1 μM insulin for 4

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h. Cells were washed twice with cold PBS and lysed with 1 ml polysome lysis buffer (20 mM HEPES pH 7.6, 5 mM MgCl2, 125 mM KCl, and 1% NP-40) supplemented with 2 mM DTT, 100 µg/ml cycloheximide, protease inhibitor cocktail (EDTA-free; Pierce), and RNasin (Thermo Scientific). After agitation for 15 min at 4°C, nuclei were pelleted (13,200 rpm for 15 min) and the protein contents of the supernatants were quantified. An equal amount of protein from each sample was fractionated by ultracentrifugation (35,000 rpm for 2 h 40 min; Beckman SW40 Ti rotor) on a 17.5–50% linear sucrose gradient. Gradients were eluted with a gradient fractionator (Brandel) and monitored with a UA-5 detector (ISCO). Fractions were prepared for protein and RNA analyses using acetone precipitation and TRIZOL, respectively.

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ACCEPTED MANUSCRIPT 2.9. Calculation of NMD efficiency Relative NMD efficiency was obtained using the following formula:

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Fold change of NMD = Test NMD/Control NMD,

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where NMD = Relative Ter mRNA/Relative Norm mRNA.

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NMD was obtained from relative PTC-containing mRNA (Ter) normalized to relative PTCfree mRNA (Norm), in which both PTC-containing and PTC-free mRNA were normalized to MUP

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mRNA as a reference.

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ACCEPTED MANUSCRIPT 3. Results and Discussion 3.1. Insulin treatment enhances NMD whereas rapamycin treatment suppresses this

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enhancement

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Insulin stimulation activates PI3K/AKT/mTOR signaling, which ultimately enhances translation and

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progression through the cell cycle (reviewed in [34]). Translation plays a pivotal role in NMD because NMD is initiated by recognition of a PTC by a scanning ribosome during translation. As inhibition of translation by cycloheximide or puromycin impairs NMD [35, 36], it is conceivable that

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increased translation would enhance NMD. To initially evaluate the effect of insulin stimulation on

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NMD upon increase of translation, HeLa cells were transiently transfected with test plasmids and a reference plasmid, where the test plasmids encoded Gl and GPx1 mRNA with either a normal

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termination codon (Norm) or a PTC (Ter) (Fig. 1A). After transfection, cells were incubated for 20 h

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and then treated with insulin under serum starvation. The level of PTC-containing mRNA was decreased by 4 h of insulin stimulation: the levels of Gl Ter and GPx1 Ter mRNA were reduced from

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15% to 11% and from 41% to 27%, respectively (Supplementary Fig. S1A). To address the effect of insulin stimulation over time course, cells were stimulated with insulin and harvested over a series of

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time points (Supplementary Fig. S1B). RT-qPCR analysis demonstrated that relative NMD levels were not changed after 3 h of insulin stimulation, but that levels decreased after 4 h of insulin stimulation. These results imply that NMD is upregulated by insulin stimulation. To determine whether insulin stimulation enhances mammalian NMD via PI3K/AKT/mTOR signaling, HeLa cells were transiently transfected with test plasmids and a reference plasmid, incubated for 20 h, and serum starved. Cells were then incubated in the presence or absence of insulin for an additional 4 h, during which the cells were treated simultaneously with rapamycin (an inhibitor of mTOR signaling) (Fig. 1B). RT-qPCR analysis demonstrated that the relative fold change of NMD without rapamycin treatment was reduced to approximately 0.6 by insulin stimulation (Fig. 1C). This finding indicates that approximately 1.7-fold (1.0 divided by 0.6) more Gl

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ACCEPTED MANUSCRIPT and GPx1 Ter mRNA was efficiently degraded after insulin stimulation compared with the nonstimulated control. Intriguingly, the cells treated with rapamycin exhibited NMD values similar

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to those of control cells (approximately 0.9 and 1.1 for Gl Ter mRNA and GPx1 Ter mRNA,

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respectively). These results indicate that NMD is enhanced by insulin-mediated stimulation of

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mTOR signaling.

3.2. Insulin stimulation augments NMD, probably via activation of translation

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Insulin activates translation by increasing the level of phosphorylated AKT, which activates mTOR

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signaling and ultimately phosphorylates eukaryotic translation initiation binding protein 4E1 (4EBP1) through a signaling cascade. To determine whether insulin-mediated translation enhancement

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upregulates NMD via AKT activation, we treated HeLa cells with a PTEN inhibitor (PTEN-I), which

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increases levels of PIP3 and phosphorylated AKT. As a control, cells were treated with wortmannin (Wort), which inhibits both PI3K and SMG1 kinase activity, the latter of which prevents UPF1

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phosphorylation and thereby blocks NMD. As an additional control, cells were treated with DMSO alone. Cells were treated with the indicated inhibitors in the presence or absence of insulin during

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serum starvation (Fig. 2A). Western blot analysis revealed that, as expected, treatment with Wort reduced the level of phosphorylated UPF1 (P-UPF1), both in the presence and absence of insulin. Furthermore, treatment with Wort reduced the amount of phosphorylated AKT (P-AKT) and 4EBP1 (P-4EBP1), indicating that Wort impairs translation. Treatment of cells with PTEN-I in the absence of insulin stimulation (Fig. 2A, lane 3) significantly increased levels of P-AKT, P-4EBP1, and PmTOR. However, in the presence of insulin stimulation, levels of P-AKT, P-4EBP1, and P-mTOR (lane 6) were only slightly increased compared with the control (lane 4) and the level of P-UPF1 remained unchanged. Together, these results suggest that inhibiting PTEN activity leads to an increase in translation without disturbing the level of P-UPF1.

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ACCEPTED MANUSCRIPT To investigate the effect of insulin-mediated and/or PTEN-I-mediated enhanced translation on NMD, HeLa cells were transiently transfected with test plasmids/reference plasmid and then

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incubated for 24 h. Transiently transfected cells were treated with the indicated inhibitors as

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described in Fig. 2A. As expected, Wort-treated cells exhibited reduced NMD as evidenced by an

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increase in the relative amount of Gl and GPx1 Ter mRNA (approximately 2.1-fold and 1.7-fold, respectively) in the absence of insulin stimulation (Fig. 2B). Similarly, NMD of Gl and GPx1 Ter mRNA was decreased by 1.7-fold and 1.6-fold, respectively, by Wort treatment in the presence of

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insulin stimulation. However, the relative NMD for both Gl and GPx1 Ter mRNA was enhanced by

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PTEN-I treatment, both in the absence and presence of insulin stimulation (from 1.0 to 0.6 and 0.2, respectively, for Gl Ter mRNA and from 1.0 to 0.5 and 0.2, respectively, for GPx1 Ter mRNA).

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Using the same lysates as in Fig. 2A, endogenous NMD targets including TBL2, GADD45B,

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MAP3K, and BAG1 were examined (Fig. 2C). This experiment confirmed that PTEN-I enhanced NMD up to two-fold compared with DMSO treatment, suggesting that decay of endogenous NMD

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targets is also controlled by PI3K/AKT/mTOR signaling. To determine whether insulin stimulation upregulates translation, we transiently co-transfected

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HeLa cells with the Gl Norm plasmid expressing the Renilla luciferase gene at the N-terminus and the pGL3 reference plasmid, which has a firefly luciferase reporter, and then measured relative Renilla luciferase activity (Supplementary Fig. S2A). As expected, insulin stimulation increased the relative translation efficiency of RLuc-Gl Norm 2.5-fold, suggesting that insulin stimulation effectively increased translation efficiency. As an alternative approach to confirm that insulin stimulation increases translation, polysome profiling was performed. Serum starvation reduced translation, as demonstrated by the decrease in the number of polysome peaks while insulin stimulation consistently enhanced mRNA translation, as demonstrated by the increase in the number of polysome peaks (Supplementary Fig. S2B). The relative level of RPS3 in polysome fractions (F7 to13) from serum-starved samples was increased upon insulin stimulation, which is consistent with

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ACCEPTED MANUSCRIPT the polysome peak results. Because the relative total amount of PTC-containing reporter transcripts after insulin treatment was approximately 1.5-fold less than that without insulin treatment, the

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patterns of overall relative distribution of reporter mRNAs (Gl and GPx1 Ter mRNAs) in the

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presence or absence of insulin treatment were comparable (the relative amount of total RNA

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corresponded to 100%). These data indicate that insulin stimulation, which enhances translation, also activates the NMD pathway. Notably, insulin stimulation did not enhance Gl mRNA splicing (Supplementary Fig. S3A, right panel). The level of Gl Norm pre-mRNA was not altered by insulin

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stimulation; similarly, the levels of cotransfected MUP mRNA, endogenous GAPDH pre-mRNA,

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and endogenous GAPDH mature mRNA also remained unchanged. Although high concentrations of insulin (100 mM) have been shown to enhance splicing, which leads to enhanced translation [37], a

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low concentration of insulin (1 μM) was employed throughout the present study. Furthermore, to

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verify that insulin stimulation does not restrict mRNA to the nucleus, cell fractionation was performed with or without insulin stimulation, and the amount of endogenous GAPDH mRNA in

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each fraction was quantified by RT-qPCR. Western blotting verified the integrity of the nuclear extract, as evidenced by the absence of the cytoplasmic control protein phospholipase C gamma

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(PLCγ) (Supplementary Fig. S3B, left panel). RT-qPCR using nuclear extracts indicated that the level of nuclear GAPDH mRNA (normalized to the level of U6 snRNA) was not significantly different in the presence or absence of insulin stimulation (Supplementary Fig. S3B, right panel), suggesting that insulin stimulation does not inhibit mRNA export from the nucleus to the cytoplasm.

3.3. Genetic silencing of endogenous AKT impairs NMD To ensure that the effects of PTEN-I treatment were due to specific inhibition of PTEN rather than pleiotropic effects, siRNA-mediated silencing was used to decrease the levels of endogenous PTEN and AKT. HeLa cells were transiently transfected with siRNA specific for UPF1, PTEN, AKT, or with nonspecific control siRNA without serum starvation. Cells were then transfected with NMD test

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ACCEPTED MANUSCRIPT and reference plasmids in the presence of serum. Western blot analysis confirmed successful siRNAmediated silencing of each target (UPF1, PTEN, and AKT) and demonstrated that this silencing did

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not affect the levels of other proteins (Fig. 2D). siRNA-mediated silencing of PTEN significantly

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increased the relative level of P-AKT, but only slightly increased the levels of P-4EBP1 and P-

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mTOR. Similarly, siRNA-mediated silencing of AKT reduced the level of P-4EBP1, suggesting that silencing of PTEN or AKT enhances or represses translation, respectively. Although PTEN knockdown increased P-mTOR and P-4EBP1 (Fig. 2D, lane 3), AKT knockdown did not

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significantly decrease the level of P-mTOR (lane 4). However, AKT knockdown did reduce the level

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of P-4EBP1 (Fig. 2D, lane 4). One possible explanation for this slight change in the level of PmTOR is that the siRNA-mediated knockdown was performed without serum starvation and

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unknown factors in the serum may have maintained the level of mTOR phosphorylation. These

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results prompted us to test whether NMD is affected by silencing of PTEN and/or AKT. To this end, cells were transfected with control, UPF1-, PTEN-, or AKT-siRNA and then retransfected with

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NMD test and reference plasmids. The relative amounts of Gl and GPx1 mRNA were quantified and used to determine the relative NMD values (Fig. 2E). UPF1 knockdown decreased the relative NMD

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by approximately 3.3-fold and 4.0-fold for Gl and GPx1 reporters, respectively. Intriguingly, PTEN knockdown increased the relative NMD approximately 2-fold for both Gl and GPx1 mRNA; on the other hand, AKT knockdown inhibited NMD by approximately 2.5-fold and 2.3-fold for Gl and GPx1 Ter mRNA, respectively. Knockdown of PTEN and AKT exerted similar effects on the abundance of endogenous NMD targets; however, the magnitude of the effect varied according to the mRNA target (Fig. 2F). One possible mechanism is that insulin stimulation or AKT knockdown indirectly increases or decreases NMD, respectively, via inhibition of transcription. To test this, we compared the stability of endogenous NMD target mRNAs after insulin stimulation and treatment with DRB, RNA polymerase inhibitor at various time points (Supplementary Fig. S4A). RT-qPCR results

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ACCEPTED MANUSCRIPT demonstrated that insulin stimulation decreased the half-lives of endogenous NMD substrates. Furthermore, both Gl and GPx1 Ter mRNAs were approximately 3-fold more stable in AKT-

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knockdown cells that were transiently transfected with NMD test and reference plasmids as

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demonstrated by RT-qPCR (Supplementary Fig. S4B). These observations are consistent with the

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results shown in Fig. 2 and support the hypothesis that increased translation enhances NMD through PI3K/AKT/mTOR signaling.

We initially evaluated whether insulin stimulation enhances NMD after siRNA-mediated

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knockdown of three NMD factors: SMG1, UPF1, and UPF2. After siRNA transfection, cells were

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transiently transfected with NMD test and reference plasmids either with or without insulin stimulation and with serum starvation (Supplementary Figs. S5A and S5B). Western blot analysis

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revealed that siRNA-mediated silencing of SMG1, UPF1, and UPF2 effectively reduced the levels of

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these proteins (Supplementary Fig. S5A). As expected, RT-qPCR results demonstrated that insulin stimulation did not upregulate NMD in SMG1-, UPF1-, or UPF2-knockdown cells (Supplementary

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Fig. S5B). These results indicate that insulin-mediated upregulation of NMD is dependent on SMG1, UPF1, and UPF2. Mammalian NMD can remove PTC-containing transcripts that do not retain an

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EJC downstream PTC, a process known as “failsafe” NMD [30, 38, 39]. Increases in EJC-dependent NMD related to insulin stimulation prompted us to test whether insulin could also boost failsafe NMD. For this, we used an intronless NMD test plasmid lacking intron 2 downstream of the PTC (Supplementary Fig. S5C) in the presence or absence of insulin stimulation. RT-qPCR analysis demonstrated that insulin stimulation did not increase failsafe NMD, but rather slightly decreased it (Supplementary Fig. S5D). We conclude that insulin-mediated enhancement of translation does not augment failsafe NMD.

3.4. Insulin stimulation remodels UPF1-bound mRNP and facilitates eIF4E-dependent NMD

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ACCEPTED MANUSCRIPT UPF1 preferentially binds PTC-containing transcripts compared with PTC-free transcripts [24, 32]. To initially evaluate whether insulin stimulation remodels UPF1-bound mRNP, UPF1-bound

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transcripts were immunoprecipitated using anti-UPF1 antibodies or normal rabbit serum (NRS) as a

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control. Immunoprecipitation (IP) was performed from lysates of cells that had been transfected with

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NMD test and reference plasmids in the absence or presence of insulin stimulation and with serum starvation. Western blot analysis of the IP inputs and eluates demonstrated that UPF1 was immunoprecipitated only by anti-UPF1 antibody and not by NRS; moreover, β-actin was not

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immunoprecipitated by anti-UPF1 antibody, demonstrating the specificity of the IP (Fig. 3A). Next,

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IP eluates were analyzed by RT-qPCR to quantify the level of UPF1-bound transcripts (Fig. 3B). Consistent with previous results [24, 32, 40], RT-qPCR indicated that approximately 1.6-fold more

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Gl Ter mRNA and 1.4-fold more GPx1 Ter mRNA coimmunoprecipitated with UPF1 compared with

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their PTC-free counterparts in the absence of insulin stimulation (Fig. 3B, left panel). Intriguingly, insulin stimulation significantly increased the amount of coimmunoprecipitated Gl and GPx1 Ter

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mRNA by approximately 3.4-fold and 3.1-fold, respectively (Fig. 3B, left panel). The relative ratios of the mRNA levels after IP (+IP) to before IP (-IP) in the absence or presence of insulin stimulation

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demonstrated that the +IP: -IP ratio of Gl and GPx1 Ter mRNA in the absence of insulin stimulation was increased by approximately 22-fold and 8.4-fold compared with their counterparts (Fig. 3B, right panel). Of note, the Gl and GPx1 Ter mRNA ratios were increased by approximately 57-fold and 45-fold, respectively, upon insulin stimulation. These results indicate that enhancement of translation by insulin stimulation increases the amount of UPF1-bound PTC-containing NMD target transcripts. To investigate whether insulin stimulation remodels UPF1-transcript binding, HeLa cells were transiently transfected with FLAG-tagged UPF1. Cells were then subjected to serum starvation in either the absence or presence of insulin stimulation. IP was performed with anti-FLAG antibodies or mouse IgG (mIgG) as a negative control (Fig. 3C). Western blot analysis of IP inputs and eluates

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ACCEPTED MANUSCRIPT indicated that FLAG-UPF1 was efficiently and comparably immunoprecipitated by the anti-FLAG antibodies. The amounts of UPF2 and CBP80 that coimmunoprecipitated with FLAG-UPF1 were

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comparable regardless of insulin stimulation; similarly, the levels of coimmunoprecipitated poly(A)

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binding protein (PABP) and eIF3b remained unchanged. Intriguingly, more eIF4E was

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coimmunoprecipitated with FLAG-UPF1, presumably through mRNA, upon insulin stimulation compared with in the absence of insulin stimulation. Of particular note, the translation of CBCbound transcripts is not inhibited by serum starvation; however, steady-state translation is inhibited

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by serum starvation and is accompanied by a decrease in 4EBP1 phosphorylation [8]. In accordance

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with previous results [41], DCP1a was coimmunoprecipitated with FLAG-UPF1, which was upregulated upon insulin stimulation. Taken together with previous reports and our observations

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indicate that insulin stimulation increased the decay of eIF4E-bound transcripts and that insulin-

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mediated enhancement of translation might maintain the interaction between UPF1 and PTCcontaining eIF4E-bound transcripts. Our results do not, however, provide direct evidence that insulin

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stimulation enhances only degradation of eIF4E-bound transcripts by NMD, and UPF1-binding transcripts do not solely represent endogenous NMD targets. Indeed, recent reports have revealed

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that NMD occurs during steady-state translation [4, 5], a finding that supports our results. To confirm that insulin stimulation enhances eIF4E-dependent NMD, we employed a dominant-negative 4EBP1 mutant (4EBP1-TTAA), in which two threonine amino acid residues (T37 and T46) were substituted with alanine residues, resulting in the inhibition of eIF4E-dependent translation. HeLa cells were transiently cotransfected with HA-tagged 4EBP1-TTAA and NMD test or reference plasmids followed by serum starvation in either the absence or presence of insulin stimulation. Western blot analysis demonstrated that the production of 4EBP1-TTAA was comparable in the presence or absence of insulin stimulation (Fig. 3D). The overexpression of 4EBP1-TTAA inhibited NMD in Gl and GPx1 mRNA by approximately 1.9-fold and 2.0-fold, respectively, in the absence of insulin stimulation (Fig. 3E). However, upon insulin stimulation,

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ACCEPTED MANUSCRIPT NMD in Gl and GPx1 Ter mRNA was inhibited 4.5-fold (from 0.6 to 2.7) and 4.3-fold (from 0.6 to 2.6), respectively, in comparison. These data most likely suggest that 4EBP1-TTAA more sensitively

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impaired eIF4E-bound NMD in the presence of insulin stimulation than its counterparts, in the

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absence of insulin stimulation. To ascertain whether overexpression of HA-4EBP1-TTAA did not

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affect the pioneer round of translation, the experiments in Fig. 3D and 3E were performed in the absence of serum depletion and insulin stimulation, and coimmunoprecipitation using anti-CBP80 antibody was performed. Western blotting analysis demonstrated that a comparable amount of

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CBP80 was specifically immunoprecipitated (Supplementary Fig. S6A). Notably, the relative

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amounts of coimmunoprecipitated Gl and GPx1 Norm and Ter mRNA were comparable in the presence or absence of HA-4EBP1-TTAA (Supplementary Fig. S6B), suggesting that overexpression

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of HA-4EBP1-TTAA did not affect the binding of CBP80 to NMD reporter transcripts. CBP80

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directly or indirectly interacts with UPF1 during the pioneer round of translation (reviewed in [3] and references therein). To determine whether overexpression of HA-4EBP1-TTAA disrupts CBP80-

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UPF1 interaction, HeLa cell lysates that were transiently cotransfected with FLAG-UPF1 and HA4EBP1-TTAA were immunoprecipitated using anti-FLAG beads. WB analysis demonstrated that the

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amount of CBP80 that coimmunoprecipitated with FLAG-UPF1 was similar in the presence or absence of HA-4EBP1 (Supplementary Fig. S6C), suggesting that overexpression of HA-4EBP1TTAA did not impair CBP80-UPF1 mRNP interaction.

3.5. Insulin stimulation induces colocalization of mCherry-DCP1a with eIF4E NMD target mRNA and decay factors including DCP1a, eIF4E, and UPF1 concentrate in cytoplasmic foci called processing bodies (PBs) [42-47]. In particular, PTC-containing mRNA and UPF1 are enriched in PBs when NMD is blocked [16, 48-50]. However, PBs do not seem to be sites of RNA decay, but rather storage sites of decay factors, because PBs are not required for mammalian NMD and a PTC-containing NMD target almost completely disappeared immediately after its export

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ACCEPTED MANUSCRIPT from the nucleus to the cytoplasm [51, 52]. Because we observed increased coimmunoprecipitation of DCP1a and eIF4E with FLAG-UPF1 after insulin stimulation, we determined whether UPF1 and

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eIF4E colocalize to PBs as a consequence of the increase of NMD induced by insulin stimulation. To

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assess this, HeLa cells were transiently transfected with a construct driving the expression of

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mCherry-tagged DCP1a (mCherry-DCP1a) and then stimulated with insulin under serum starvation conditions. Cells were immunostained with anti-UPF1, anti-CBP80, and anti-eIF4E antibodies. Consistent with previous reports [53, 54], UPF1 and eIF4E were mainly cytoplasmic in either the

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absence or presence of insulin stimulation, although a small proportion of each was nuclear (Fig. 4A).

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In contrast to the predominantly cytoplasmic localization of UPF1 and eIF4E, CBP80 was mainly observed in the nucleus. Of note, the predominant localization of each protein and the number of PBs

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were unchanged by insulin stimulation. Pearson’s correlation coefficient was used to quantify the

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fluorescence signal to determine whether insulin stimulation induces colocalization of mCherryDCP1a with UPF1, CBP80, or eIF4E (Fig. 4B). The amount of CBP80 that colocalized with

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mCherry-DCP1a was not significantly altered by insulin stimulation. Surprisingly, the amount of mCherry-DCP1a that colocalized with UPF1 and eIF4E was increased approximately 1.7-fold and

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2.0-fold, respectively, by insulin stimulation. These results suggest that although PBs are not required for NMD, insulin stimulation augments the colocalization of UPF1 or eIF4E in PBs, probably as products of increased NMD. Considering all of our observations, it is plausible that eIF4E-bound NMD targets are removed upon insulin stimulation via PI3K/AKT/mTOR signaling, as suggested in Fig. 5. Our results complement the current NMD model by describing the role of NMD in the degradation of eIF4E-bound mRNA and also raise the possibility that extracellular signaling can regulate NMD.

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ACCEPTED MANUSCRIPT Acknowledgements We would like to thank Lynne E. Maquat (University of Rochester) for providing the NMD test and

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reference plasmids, pACTAG2-HA-4EBP1-TTAA, and anti-UPF1 antibody and Yoon Ki Kim

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(Korea University) for providing the anti-P-UPF antibody. This research was supported by the Basic

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Science Research Program through the National Research Foundation of Korea, which is funded by the Ministry of Education, Science and Technology (2015R1D1A1A01058878 to J.H.) and by a

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Foundation of the Korean government (MSIP).

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grant from the Medical Research Center (2011-0028261 to J.H.) funded by the National Research

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Insulin stimulation upregulates NMD, whereas rapamycin blocks this effect. (A) Schematic

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representation of spliced NMD Gl and GPx1 mRNA reporter transcripts. Boxes denote exons. Norm

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Ter and PTC represent a normal termination codon and a premature termination codon, respectively.

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(A)n, poly(A) tail. (B) Schematic representation of the experimental workflow, including transfection, serum starvation, insulin stimulation, and rapamycin treatment. Briefly, HeLa cells were transiently transfected with NMD test and reference plasmids. Cells were then incubated for 20 h in the

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presence of serum and then subjected to serum starvation for an additional 24 h, after which the cells

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were treated with or without insulin (1 μM) and/or rapamycin (100 nM) for 4 h. (C) The relative amounts of Gl (top panel) and GPx1 (bottom panel) mRNA from cell lysates in (B) were assessed by

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RT-qPCR. Relative NMD is expressed as the ratio of the level of Ter mRNA to the level of Norm

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mRNA, each of which was normalized to MUP reference mRNA as described in Materials and Methods. The level of NMD in control cells that were not treated with either insulin or rapamycin

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was set to 1.0. Mean values and standard errors were calculated from at least four independent experiments. The number above each bar represents the average. Asterisks denote statistically

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significant differences (**, p <0.05; unpaired Student’s t-test).

Fig. 2. Inhibition or siRNA-mediated knockdown of PTEN upregulates NMD. (A) Western blot analysis of lysates from HeLa cells treated with wortmannin (Wort), PTEN inhibitor (PTEN-I), or DMSO (control) under serum starvation conditions in the absence or presence of 1 μM insulin. (B) As in (A); however, cells were transiently transfected with NMD test and reference plasmids, and RT-qPCR was performed. Normalized levels of Gl and GPx1 mRNA obtained from DMSO (control)-treated cells were set to 1.0. (C) As in (A); however, the levels of endogenous NMD targets were quantified by RT-qPCR. The level of NMD target mRNA was normalized to that of GAPDH mRNA. The normalized level of each NMD target mRNA from DMSO-treated cells (control) was

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ACCEPTED MANUSCRIPT set to 1.0. C, W, and P indicate control, wortmannin, and PTEN-I treatments, respectively. (D) Efficiency of siRNA-mediated knockdown as shown by Western blot analysis of lysates from HeLa

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cells transfected with the specified siRNA sequences. (E) As in (D); however, cells were transiently

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transfected with NMD test and reference plasmids after siRNA transfection. RT-qPCR was

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performed to quantify the relative levels of Gl and GPx1 mRNA. (F) As in (D); however, the level of endogenous NMD target mRNA was quantitated by RT-qPCR. GAPDH mRNA served as an internal control. The normalized NMD level of each target mRNA from control siRNA-transfected cells was

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set to 1.0. C, U, P, and A indicate transfection with control, UPF1, PTEN, and AKT siRNA,

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respectively. The four left lanes represent 3-fold dilution of cell lysates in (A) and (D), demonstrating that WB conditions were semiquantitative. Mean values (%) with standard deviations

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(S.D.) were calculated from at least three independent experiments in (A) and (D). Columns and

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error bars indicate the means and standard errors of at least three independent experiments in (B), (C), (E) and (F). Numbers above the error bars indicate the mean values from these experiments. **, p

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<0.01 by unpaired Student’s t-test.

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Fig. 3. Insulin stimulation remodels UPF1-bound mRNP, presumably in eIF4E-bound NMD transcripts. (A) Lysates from HeLa cells that were transiently transfected with NMD test and reference plasmids in the absence or presence of insulin stimulation with serum starvation were subjected to immunoprecipitation (IP) using anti-UPF1 antibodies or normal rabbit serum (NRS) as a control for nonspecific interactions. IP inputs and eluates were analyzed by Western blotting. (B) RT-qPCR was performed using the lysates from (A). The levels of Gl and GPx1 mRNA were normalized to the levels of MUP mRNA in the IP inputs and eluates as appropriate. (Left panel) The normalized levels of Gl and GPx1 Norm mRNA in each IP were set to 100. (Right panel) The normalized levels of Gl and GPx1 mRNA in the IP eluates are expressed as ratios of the normalized levels of Gl and GPx1 mRNA in the IP inputs. (C) HeLa cells that were transiently transfected with

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ACCEPTED MANUSCRIPT FLAG-UPF1 were treated with or without insulin under serum starvation conditions. Cell lysates were generated and IP was performed with anti-FLAG antibodies or mouse IgG (mIgG) as a

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negative control. IP inputs and eluates were analyzed by Western blotting. The four left lanes

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represent 3-fold dilution of cell lysates, demonstrating that WB conditions were semiquantitative. (D)

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HeLa cells were transiently transfected with NMD test and reference plasmids, and a dominantnegative mutant, HA-tagged unphosphorylated 4EBP1 (HA-4EBP1-TTAA) in the presence or absence of insulin stimulation with serum starvation. Western blot analysis indicated the expression

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of HA-4EBP1-TTAA. (E) RT-qPCR using cell lysates in (D) was performed to quantify the relative

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amounts of Gl and GPx1 mRNA, where mRNA was normalized to MUP mRNA. The ratios with empty vector transfection (-) in the absence of insulin stimulation were set to 1.0. The columns and

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error bars indicate the mean and standard error of at least three independently performed experiments.

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unpaired Student’s t-test.

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Numbers above the error bars indicate the mean values. **p <0.01 in (B) and (E) as determined by

Fig. 4. Insulin stimulation induces the colocalization of mCherry-DCP1a with UPF1 and eIF4E. (A)

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HeLa cells were transiently transfected with mCherry-DCP1a and stimulated with or without insulin treatment under serum starvation conditions. Immunofluorescence analysis was performed using rabbit anti-UPF1, rabbit anti-CBP80, or mouse anti-eIF4E antibodies. Primary antibodies were visualized by incubation with secondary AlexaFluor 488 goat anti-rabbit or anti-mouse IgG antibodies as appropriate. (B) Colocalization of mCherry-DCP1a with UPF1, CBP80, and eIF4E was quantified using Pearson’s correlation coefficient analysis in Image J (JACoP) software (n>8). Columns and error bars express the mean and standard errors. **, p <0.05 by unpaired Student’s ttest (ns, not significant).

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ACCEPTED MANUSCRIPT Fig. 5. Suggested model for insulin-mediated enhancement of NMD. PTC-containing CBP20/CBP80-bound transcripts, which are bound to UPF1 [21], are exported from the nucleus to

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the cytoplasm. CBP20/CBP80-bound NMD targets are then degraded in either an EJC-dependent or -

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independent manner [55]. The cap-binding heterodimer CBP20/CBP80 is replaced by eIF4E, with

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the help of karyopherin importin β (IMPβ) [56]. Bound UPF1 on the coding region of the transcript is removed by a translating ribosome. Translation is inhibited with the 4EBP-eIF4E complex at the cap. The interaction of eIF4G-eIF4E resulting from phosphorylation of 4EBP, which is induced by

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insulin-mediated PI3K/AKT/mTOR signaling, initiates translation. This, in turn, triggers the

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degradation of PTC-containing eIF4E-bound transcripts.

Fig. S1. Insulin stimulation increases the efficiency of NMD over time. (A) HeLa cells were

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transiently transfected with NMD reporter and reference plasmids in the presence or absence of 1 μM insulin for 4 h under serum starvation and RT-qPCR was performed to quantify the amounts of Gl

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and GPx1 mRNA. The relative level of PTC-free transcripts (Norm) was set to 100%. Columns and error bars express the mean and standard error. **, p <0.01 by unpaired Student’s t-test. (B) As in

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(A); however, cells were harvested at the indicated time points and the amounts of Gl and GPx1 mRNA were quantified by RT-qPCR. The NMD value represents the ratio of the level of Ter mRNA to the level of Norm mRNA, where each mRNA level was normalized to that of MUP mRNA (a reference) as described in the Materials and Methods. The level with insulin stimulation at 0 h was set to 1.0. Mean values and standard errors were calculated from four independent experiments.

Fig. S2. Insulin stimulation upregulates translation. (A) HeLa cells were transiently transfected with NMD Gl reporter (RLuc-Gl), which contains the Renilla luciferase reporter gene at the N-terminus, and the reference plasmid expressing firefly luciferase (FLuc), followed by incubation in the presence or absence of insulin stimulation with serum starvation. Cell lysates were tested for

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ACCEPTED MANUSCRIPT translation efficiency, which was calculated by the relative ratio of RLuc activity to FLuc activity normalized by the relative ratio of RLuc mRNA to FLuc mRNA. The ratios in the absence of insulin-

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stimulation were set to 1.0. Columns and error bars indicate the means and standard errors of at least

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three independent experiments. The numbers above the error bars indicate mean values from the

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experiments. **, p <0.05 by unpaired Student’s t-test. (B) HeLa cells that had been transiently transfected with NMD test and reference plasmids were stimulated with insulin after serum starvation for 24 h. Polysome profiling analyses were performed to monitor global translation status

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with cell extracts with or without insulin stimulation. Polysome peaks and the distribution of RPS3

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using Western blot analyses were assessed to compare global translation. Total RNA was prepared from each fraction and the distribution of the relative levels of each mRNA (Gl Ter and GPx1 Ter

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mRNA normalized to MUP mRNA) was monitored using RT-qPCR. The relative overall distribution

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of Gl or GPx1 Ter mRNA in the presence or absence of insulin stimulation at each fraction was

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assessed.

Fig. S3. Insulin stimulation does not affect splicing or mRNA export. (A) (Left panel) Diagram of Gl

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and GAPDH pre-mRNA, where blank boxes denote exons and lines denote introns. Arrowheads represent the primers used to generate products of Gl and GAPDH pre-mRNA in the semiquantitative RT-PCR (RT-sqPCR). (Right panel) HeLa cells were transiently transfected with Gl Norm and MUP plasmids. After one day of incubation, cells were serum-starved for 24 h and incubated in the absence or presence of insulin stimulation. The indicated mRNAs were observed by RT-sqPCR. (B) HeLa cells were treated with or without insulin stimulation after a 24-h period of serum starvation. Nuclear and cytoplasmic fractions were then generated from cell lysates. (Left panel) Nuclear and cytoplasmic fractions were subjected to Western blotting using anti-PLCγ and anti-p62 antibodies as cytoplasmic and nuclear markers, respectively. Three-fold dilutions of total cell lysates were loaded in the four leftmost lanes, demonstrating that the Western blotting conditions

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ACCEPTED MANUSCRIPT were semiquantitative. (Right panel) The level of GAPDH mRNA was normalized to the level of U6 snRNA in the nuclear lysates by RT-qPCR. The normalized level of GAPDH mRNA in the absence

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of insulin stimulation was set to 1.0. Columns and error bars indicate the means and standard errors

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of at least three independent experiments (ns, not significant).

Fig. S4. Insulin stimulation and depletion of AKT impair NMD. (A) After one day of incubation with serum, cells were starved for 24 h and then incubated in the presence or absence of insulin with

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100 μg/ml 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). The normalized levels of

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endogenous NMD target mRNAs to GAPDH mRNA were plotted as a function of time after DRB treatment. Means and standard errors were calculated from at least six independently performed

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transfections and RT-qPCR. t1/2, half-life. (B) As in (A); however, HeLa cells were transiently

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transfected with NMD test and reference plasmids. After one day of incubation, cells were starved for 24 h and then incubated in the presence or absence of insulin for 4 h. Cells were treated with

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DRB for 20 min. The levels of Gl and GPx1 mRNA were obtained by RT-qPCR and normalized to the level of MUP mRNA. Columns and error bars represent the means and standard errors of at least

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three independent experiments. **, p <0.05 as determined by unpaired Student’s t-test.

Fig. S5. Insulin stimulation does not rescue NMD in SMG1-, UPF1-, or UPF2-knockdown cells, or EJC-independent NMD. (A) Western blot analysis of transient siRNA-mediated knockdown efficiency in HeLa cells with the designated NMD factors. Transiently transfected cells were incubated in the presence or absence of insulin stimulation with serum starvation. (B) As in (A); however, cells were transiently transfected with NMD test and reference plasmids after siRNA transfection. RT-qPCR was used to quantify the relative mRNA levels of the designated genes. The normalized levels of Gl and GPx1 mRNA obtained from control siRNA-transfected cells without insulin stimulation were set to 1.0. (C) Schematic representation of the two pmCMV-Gl constructs

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ACCEPTED MANUSCRIPT (with and without intron 2). Blank boxes and lines represent exons and introns, respectively. (D) HeLa cells were transiently transfected with either pmCMV-Gl or the version lacking intron 2, in

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addition to the reference plasmids described in (C). Cells were then stimulated with insulin under

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serum starvation, and RT-qPCR was used to quantify the relative amounts of Gl mRNA. The level of

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Gl mRNA was normalized to that of MUP mRNA. The ratios in the absence of insulin stimulation for each transfection were set to 1.0. The columns and error bars indicate the mean and standard errors of at least three independently performed experiments. Numbers above the error bars indicate

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the mean values. **p <0.05 as determined by unpaired Student’s t-test (ns, not significant).

Fig. S6. Overexpression of HA-4EBP1-TTAA does not affect the features in pioneer round of

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translation. (A) HeLa cell lysates that were transiently cotransfected with NMD reporters and HA-

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tagged 4EBP1-TTAA were immunoprecipitated using anti-CBP80 antibody or rabbit IgG (rIgG). IP inputs and eluates were analyzed by Western blotting. (B) RT-qPCR was performed using the lysates

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from (A). The levels of Gl and GPx1 mRNA were normalized to the level of MUP mRNA. The normalized levels of Gl and GPx1 mRNA in the cell lysates transfected with empty vector (control)

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were set to 100%. The columns and error bars indicate the mean and standard errors of at least three independently performed experiments (ns, not significant). (C) HeLa cell lysates that were transiently cotransfected with FLAG-UPF1 and HA-4EBP1-TTAA (+) or empty vector (-) as a control were immunoprecipitated with anti-FLAG antibody-conjugated beads or mIgG. IP inputs and eluates were analyzed by Western blotting.

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ACCEPTED MANUSCRIPT Table S1. siRNA and primer sequences.

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Forward (5’ to 3’)

Reverse (5’ to 3’)

ACCATTGTTCACAGGCAAGAGCAG TCACCATTCACCTCGCACTTCTCA TCCTGGTGAGAAGTCTCC TATTGTTTCTGCTTCTTGGAT TCTTATTAATTCGCAAACTGG GGTTCAGACATTGCAAGGGG TGTTCTGCTCCACTGTGTCAC CAAGATCATCAGCAATGCC

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TGCACGTGGATCCTGAGAACTTCA CGGTTTCCCGTGCAATCAGTTCGG CTGATGGGGCTCTATG GCAGTCATTTACCACATGC GAGTGAGACTGACTGCAAGC GGCCCGTGTGTGTTGGAAGGG AAGATGGTTGCCGGGTCATG CTGTGGTCATGAGTCCTTCC TCGCTTCGGCAGCACATATAC

TGCGTGTCATCCTTGCGCAG

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qPCR primers β-Gl GPx1 MUP TBL2 GADD45B MAP3K14 BAG1 GAPDH snU6

Antisense (5’ to 3’) AAUGGAGCGGAACUGCAUCUUGG GAUGGCUGGGACAAAAGCC UACUUUGGCGCACAUACACUU AUAGUUUCAAACAUCAUCUUG UCCUGGUUGUAGAAGGGCA UGAUUUUGCAUCUGAAGAG

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Sense (5’ to 3’) CCAAGAUGCAGUUCCGCUCCAUU GGCUUUUGUCCCAGCCAUC AAGUGUAUGUGCGCCAAAGUA CAAGAUGAUGUUUGAAACUAU UGCCCUUCUACAACCAGGA CUCUUCAGAUGCAAAAUCA

Forward (5’ to 3’) GCCTATTGGTCTATTTTCCC ACCCCCATAGGCGAGATCCC

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sqPCR primers Gl pre-mRNA GAPDH pre-mRNA

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siRNA UPF1 UPF2 SMG1 PTEN AKT1/2 AKT3

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Reverse (5’ to 3’) GAGGAGGGGAAGCTGATATC CTGCACTCACCCCAGCCTTC

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 Activation of PI3K/AKT/mTOR signaling by insulin upregulates NMD

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 Insulin stimulation increases the binding of UPF1 to PTC-containing transcripts

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 Insulin stimulation augments the binding of UPF1 to eIF4E-bound transcripts

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 The colocalization of UPF1 and eIF4E in P-bodies is increased by insulin stimulation

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