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Molecular Cell, Volume 50 Supplemental Information Akt-Mediated Phosphorylation of Argonaute 2 Downregulates Cleavage and Upregulates Translational Repression of MicroRNA Targets Shane R. Horman, Maja M. Janas, Claudia Litterst, Bingbing Wang, Ian J. MacRae, Mary J. Sever, David V. Morrissey, Paul Graves, Biao Luo, Shaikamjad Umesalma, Hank H. Qi, Loren J. Miraglia, Carl D. Novina, and Anthony P. Orth

Document S1. Figures S1–S5, Table S2, and Supplemental

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Page 1: Document S1. Figures S1–S5, Table S2, and Supplemental

Molecular Cell, Volume 50

Supplemental Information

Akt-Mediated Phosphorylation of Argonaute 2

Downregulates Cleavage and Upregulates

Translational Repression of MicroRNA Targets

Shane R. Horman, Maja M. Janas, Claudia Litterst, Bingbing Wang, Ian J. MacRae, Mary J. Sever, David V. Morrissey, Paul Graves, Biao Luo, Shaikamjad Umesalma, Hank H. Qi, Loren J. Miraglia, Carl D. Novina, and Anthony P. Orth

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Figure S1. Description and Validation of the Human Kinome RNAi Screen

(A) Schematic representation of the FLuc (D1) shRNA target site within the coding region (ORF) of the firefly luciferase gene (FLuc). (B) D1 perfect match shRNA does not affect Luc mRNA levels. Analysis of Luc mRNA and Luc protein from single cell sorted HeLa-Luc/FLuc D1 shRNA clones. qRT-PCR of Luc mRNA transcripts using primers flanking the supposed cleavage site reveal an absence of target cleavage. (C) Ago2 is the only participating Ago protein in shRNA-mediated silencing of luciferase in D1 cells. (D) Representative western blots confirming Ago knockdowns. (E) Representative western blots confirming TRBP and Dicer knockdowns. (F) Representative western

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blots confirming Akt and MAPKAPK2 (MK2) knockdowns. Note that MK2 knockdown was not as efficient as knockdown of the Akt isoforms. Error bars indicate SD. ***P ≤ .001.

Figure S2. Purity of Proteins Used for Kinase Assay

Coomassie-stained acrylamide gel of 20 µg purified Ago2 (WT and S387A), Akt3 (WT and T305A), Akt2, Akt1 and Histone H2B proteins used in kinase assay. No other co-purifying protein bands were detectable on gel.

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Figure S3. Akt Does Not Affect Ago2 Loading but Positively Regulates Repression Mediated by Various miRNAs

(A) Western analyses of FLAG-Ago2 cDNA construct expression in HeLa and HeLa-D8 cells. (B) Ago2 S387 phosphorylation status does not affect Ago2 loading of the exogenous CXCR4 siRNA or endogenous miRNAs. HeLa cells were co-transfected with the CXCR4 siRNA and either the negative control empty plasmid (FLAG-vector) or plasmids expressing WT or Ago2-S387 mutants; after 24 hrs anti-FLAG IP was performed. The levels of the guide strand of the CXCR4 siRNA or guide strands of indicated endogenous miRNAs were assessed by northern blotting and normalized to tRNA. The IP efficiency was confirmed by anti-FLAG western blotting and normalized to GAPDH. (C) Antagonizing miR-21 activity leads to increased protein but not mRNA levels of a firefly luciferase miR-21 reporter in HeLa-D8. HeLa-D8 cells that express a firefly luciferase reporter with six miR-21 binding sites in the 3’UTR were transfected with antagomir-21 and luciferase protein and mRNA levels were monitored for 6 days. Luc protein was quantified by luciferase assay and mRNA was quantified by qPCR. (D) Loss of repression after Akt3 knockdown can be rescued by both WT and S387 mutant Ago2. HeLa-D8 cells were first transfected with siRNAs against endogenous Akt3 and Ago2 or a non-targeting

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control siRNA and after 24 hours transfected with expression vectors containing cDNAs encoding Ago2-WT, Ago2-S387A or Ago2-S387E. After a further 24 hours, cells were subjected to a luciferase assay. (E) Western analyses of Akt isoform knockdown in HEK293T cells. (F) Akt knockdowns do not affect Ago2 loading of the exogenous CXCR4 siRNA or endogenous miRNAs. HeLa cells were first transfected with the negative control siRNA (siNT) or siRNAs against indicated kinases, and after 48 hrs were co-transfected with the CXCR4 siRNA and the plasmid expressing wildtype (WT) Ago2. After 24 hrs, anti-FLAG IP was performed. The levels of the guide strand of the CXCR4 siRNA or guide strands of indicated endogenous miRNAs were assessed by northern blotting and normalized to tRNA. The IP efficiency was confirmed by anti-FLAG western blotting and normalized to GAPDH. (G) Positive effects of AKT on miRNA-mediated repression are not miRNA-specific. HeLa cells were transfected with the negative (siNT) or positive (siAgo2) control siRNAs, a pool of let-7 antagomirs (anti-let7a, c, f, and g), or siRNAs against indicated kinases, and after 72 hrs protein levels of two validated let-7 targets (RAS and IMP-1) and a validated miR-21 target (PDCD4) were assessed by western blotting and normalized to GAPDH. (H) Effects of Akt knockdown on two Renilla luciferase reporters that harbor the 3’UTRs of let-7 targets (HMGA2 or KRAS) and the FL6X reporter (Figure 3A, top). Knockdown of Akt3 resulted in decreased repression of the let-7-regulated KRAS reporter as well as the FL6X reporter. (I) Screenshot of a BioGPS webpage (www.biogps.com) displaying the gene expression profile of akt3 across several different cell lines (part of the NCI60 panel). The arrow indicates akt3 expression in HEK293T cells (approximately 2 x median). Error bars indicate SD. *P ≤ .05, ***P ≤ .001.

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Figure S4. Phosphorylation of Ago2 at S387 Is Important for Efficient Association with the P Body Marker GW182

(A) Knockdown of Akt1 or Akt2 does not affect Ago2 localization to P-bodies. Immunofluorescence pictures of HeLa-EGFP-Ago2 cells harboring MAPKAPK2, Akt1 or Akt2 knockdown. Panels display representative HeLa-EGFP-Ago2 cells stained with immunofluorescent antibodies to GW182 (Alexa647-red), phosphoAgo2 (pS387; Alexa594-yellow) and Hoechst (blue); EGFP (green)

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represents endogenous Ago2. White arrows indicate Ago2 not co-localized with GW182 bodies. (B) S387 phosphorylation increases Ago2/GW182 co-localization. Immunofluorescence pictures of HeLa cells transfected with either EGFP-Ago2-WT-, EGFP-Ago2-S387A- or EGFP-Ago2-S387E cDNA expression vectors and analyzed for GW182 co-localization. Panels display representative cells stained with immunofluorescent antibodies to GW182 (Alexa647-red) and Hoechst (blue); EGFP (green) represents expressed Ago2. White arrows indicate Ago2 not coordinated with GW182 bodies. (C) Co-localization quantitation from (B) were enumerated on a confocal microscope with n = 25 cells. Error bars indicate SD. ***P ≤ .001.

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Figure S5. Akt Does Not Affect Passenger Strand Cleavage but Negatively Regulates miRNA-Directed Target mRNA Cleavage

(A) Ago2 phosphorylation by Akt3 does not affect passenger strand cleavage activity of Ago2. Purified GST-Ago2 was incubated with purified His-tagged Akt1 or Akt3 and the passenger strand cleavage

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activity of the phosphorylated Ago2 was assayed by incubating the Ago-Akt mixture with 5’-labeled double-stranded CXCR4 siRNA substrate. The cleavage products were separated on an 8% denaturing gel that was dried and exposed to a Phosphoimager. The specific cleavage products are indicated with an arrow. The cleavage efficiency (%) was quantified by the equation: (intensity of cleavage products / intensity of total input) x 100. The assay was separately performed twice yielding identical data. (B) Akt phosphorylation of Ago2 at S387 reduces target mRNA cleavage activity of Ago2 in vitro, which is partially alleviated through inhibition of Akt3 by the pan-Akt inhibitor KP372-1 (KP). Purified Ago2 WT or Ago2 S387A was incubated with purified Akt3 in the presence of ATP and either KP372-1 or a DMSO control. The target mRNA cleavage activity of the phosphorylated Ago2 was assayed by incubating the Ago2-Akt mixture with the antisense strand of CXCR4 siRNA and 5’-labeled CXCR4 substrate. Specific cleavage products are indicated with an arrow based on the ladder generated from partial digestion of input substrate by RNase T1 (T1). (C) Knockdown of Akt2, Akt3 and MK2 resulted in increased cleavage of FL1P reporter mRNA. Effects of Akt knockdown on the FL1P firefly luciferase reporter (top) that harbors one perfect CXCR4 siRNA binding site in its 3’UTR and reports target mRNA cleavage. (D) Western analyses of His-Akt expression in HeLa-D8 or A375 cells. (E) Western analyses of Akt or MK2 knockdown in HeLa-D8 cells. (F) Decreased levels of endogenous miRNA-directed cleavage targets in Akt knockdown cells are miRNA-dependent. HeLa cells were co-transfected with Akt- or MK2-specific siRNAs along with miRNA-specific antagomirs or a negative control antagomir. After 48 hrs, antagomirs were re-transfected, and RNA levels were assessed by qRT-PCR 24 hrs later. Knockdown of all Akt isoforms decreased miRNA-targeted mRNA transcript levels (grey columns) and these decreases were alleviated upon inhibition of the targeting miRNA (white columns). (G) Knockdown of Akt isoforms in A375 cells measured by qRT-PCR. Error bars indicate SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001.

Table S2. Sequences of siRNAs and shRNAs Used in this Study

Gene siRNA (Antisense) shRNA (Antisense) Akt1 CCAUAGUUGCGGGCCCGGUCC AGUGCCCUUGCCCAGCAGC Akt2 CCAAUGAAGGAGCCGUCGCUC GGGUGGCAGGAGCUUCUUC Akt3 AGAAACGUGUGCGGUCC GCUUCUGUCCAUUCUUCCC

Mapkapk2 UCGGAUGCGAGUCUUCA GGCACUGGUCAUCUCCUCC Ago1 UUUGAUCUCAAUCCCAUUG Ago2 UUUAGCAAACCAUAUUUCC Ago3 UAAAGUAACGUCUAAAUCU Ago4 UCUUGAUACAUCGCUGUCC Dicer UCCAGAGCUGCUUCAAGCA Trbp UUUCGAAGACCAUCCAGGC D1 UAAGACGACUCGAAAUC UAAGACGACUCGAAAUC NT ACGUGACACGUUCGGAGAA AUUACACUUCGGUUCCGCUU

CXCR4 UGUUAGCUGGAGUGAAAACUU Ago2 3’UTR AGUUUGAAAUCUGGGACGGAAGGCAUU

 

All siRNAs were synthesized with 5’-phosphates and RNA bases, with the exception of the 3’dTdT DNA bases that were often added to improve stability and protection from 3’ exonucleases.

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SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Real-Time Quantitative PCR

Taqman primer/probe sets for Pten (Hs00829813_s1), HoxB8 (Hs00256885_m1), Atpaf1

(Hs01103601_m1), Plekhm1 (Hs01655447_s1), Pfkfb1 (Hs00159997_m1), Lypd3 (Hs01012111_m1) and

Gapdh (Hs99999905_m1; as the endogenous internal control) were purchased from Applied Biosystems.

Western Blots

Primary antibodies used for immunoblotting were anti-Akt1 (2967), anti-Akt2 (2962), anti-Akt3

(4059), anti-MAPKAPK2 (3042), anti-FLAG (2368), anti-GAPDH (2118), anti-RAS (3965) (Cell Signaling

Technologies), anti-EIF2C1 (Ago1, ab5070), anti-EIF2C2 (Ago2, ab57113), anti-EIF2C3 (Ago3, ab3593),

anti-EIF2C4 (Ago4, ab52724), anti-Dicer (13D6, ab14601) anti-firefly luciferase (ab635-100) , anti-βactin

(ab20272-100), anti-PDCD4 (ab45124) (Abcam), anti-GW182 (sc-56314), anti-IMP1 (E-20, sc-21026),

anti-TRBP (sc-134110), anti-PTEN (sc-7974) (Santa Cruz Biotechnology Inc.) anti-PDCD4 (A301-107A,

Bethyl). Primary rabbit polyclonal antibodies and primary mouse monoclonal antibodies were detected

using HRP-conjugated goat anti-mouse IgG (Millipore) and HRP-conjugated goat anti-rabbit IgG (Abcam)

at 1:5000 dilution.

Immunofluorescence Assays

Primary antibodies used for the study were anti-GW182 (Santa Cruz Biotechnology Inc.) and anti-

phosphoAgo2 (pS387). Secondary antibodies used were Alexa Fluor® 647 donkey-anti-mouse IgG

(1:750, Invitrogen) and Alexa Fluor® 594 goat-anti-rabbit IgG (1:750, Invitrogen). Hoechst dye (1.3 µM,

Invitrogen) was used as a nuclear reference. Lasers used were: 405nm (Hoechst), 488nm (EGFP),

561nm (Alexa Fluor® 594) and 640nm (Alexa Fluor® 647) in conjunction with the appropriate filter sets.

Cloning of shRNA Lentiviral Constructs and Virus Production

The lentiviral vector (derived from LentiLox3.7, ATCC) was made Gateway adaptable and

contained a human H1 RNA polymerase-III promoter for shRNA expression. Each shRNA insert was

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designed as a synthetic duplex with overhanging ends identical to those created by restriction enzyme

digestion (BbsI at the 5' and XbaI at the 3'). The coding region for each hairpin was contained within a

single oligonucleotide (upper oligo: 5'-TTT [N19] TTCAAGAGA [antisense N19] TTTTT-3') and its

complementary equivalent (lower oligo: 5'-CTAGAAAAA [N19] TCTCTTGAA [antisense N first 18] -3'). Each

55-mer duplex comprises the shRNA encoding region (sense stem, loop sequence and anti-sense stem)

and a pol-III termination signal consisting of a run of 5 'T's. The 19-mer stem sequences were chosen

using GNF’s configurable shRNA picker software.

For lentivirus production constructs were co-transfected into HEK293T cells with packaging

vectors from the ViraPower Lentiviral Expression Kit (pMDL.gp.RRE, pRSV-REV & pCMV-VSVG;

Invitrogen) and Fugene6 (Roche) in Poly-D-Lysine coated 96-well plates or 10cm dishes. Media was

changed at 24 hrs post-transfection and viral supernatants were collected 48 hrs post-transfection. Viral

supernatants were concentrated 20 fold using Amicon Ultra Centrifugal Filter Devices (Millipore).

LC-MS

Frozen kinase reactions were quickly thawed in room temperature water, and proteins were

denatured and reduced by addition of 40 µL of 8M Urea / 0.1M Tris HCl pH8.0 / 20mM DTT. The

samples were placed in a 60˚C water bath for 20 min, and after cooling to room temperature, the

cysteines were modified by addition of 4 µL of 500mM IAM and incubated in the dark for 45min. The

samples were diluted to 160 µL and split in two. 1 µL of 0.5 µg / µL chymotrypsin or trypsin was added to

each tube. The trypsin sample was placed in a 37˚C heating block while the chymotrypsin sample was

left at room temperature overnight. Both digests were acidified to 0.1% TFA and an aliquot of each was

analyzed by LC/MS on the LTQ XL using CID and ETD.

Samples for ETD analysis were manually loaded onto a 360µm OD x 100µm ID precolumn with

2cm Monitor C18 packing and washed. The precolumn was then placed in line with a 360µm OD x 75µm

ID analytical column with 10cm of Monitor C18 packing and peptides were eluted with a 60min linear

gradient from 0% solvent B to 40% solvent B (Solvent B: 0.1M HOAC, ACN; Solvent A: 0.1M HOAc,

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water). Each digest was analyzed with an ETD only method and a CID only method. CID analyses were

performed with the aid of the autosampler for injection.

LC/MS data was searched using MASCOT against limited databases contained Ago2, Akt1, Akt2, Akt3

and approximately 165 other proteins. Variable phosphorylation on Ser, Thr, and Tyr was allowed. The

search results were compiled in Scaffold 2.0 and filtered for Min Protein: 99.9%, Min # peptides: 2, Min

Peptide: 95%. Spectra assigned to phosphopeptides were manually validated and annotated.

miRNA and siRNA Ago Loading Assays

HeLa cells were lysed for 15 min on ice in lysis buffer containing 50 mM Tris pH 7.4, 100 mM

NaCl, 0.5 mM EDTA, 1% Triton X-100, 0.4 U / µL RNase inhibitors, and protease inhibitor cocktail tablet,

and centrifuged at 12,000 x g for 15 min at 4°C. Mouse IgG agarose (Sigma) and anti-FLAG M2 agarose

(Sigma) were washed in TBS (50 mM Tris pH 7.4, 100 mM NaCl, 0.5 mM EDTA, 1% Triton X-100). After

pre-clearing lysates for 1 hr at 4°C with mouse IgG agarose, IPs were performed for 1 hr at 4°C with anti-

FLAG agarose pre-blocked with BSA and tRNA. After washing the beads five times with TBS, complexes

were eluted with 150 µg / mL FLAG peptide in lysis buffer by shaking for 30 min at 4°C. To detect

miRNAs and siRNAs, RNA was extracted with Trizol LS (Invitrogen) and resolved on 12% urea-PAGE in

1X TBE. Gels were equilibrated in 0.5X TBE and transferred onto Hybond membrane (GE Healthcare)

using a semi-dry transfer cell (BioRad). RNA was UV-crosslinked to the membrane, pre-hybrizided for 1

hr at 42°C in 5X SSPE, 2X Denhardt’s solution, and 0.1% SDS containing denatured salmon sperm DNA.

Membranes were incubated overnight at 42°C with antisense LNA and 2’OMe antagomir probes labeled

at the 5’ ends with γ-ATP. Membranes were washed for 10 min at room temperature with 2X SSC, 0.1%

SDS and for 10 min with 0.2X SSC, 0.1% SDS.

In Vitro Passenger-Strand Cleavage Assays

RNA cleavage assays were performed as described previously (Wang et al., 2009; Wang et al.,

2008). Briefly, bacterially-expressed GST-Ago was incubated with either Akt1, Akt2, Akt3, or Akt3-T305A

in the presence of ATP for 1 hour. The cleavage activity of phosphorylated Ago was assayed by

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incubating 5'-radiolabeled target substrate RNA or siCXCR4 (5'-radiolabeled anti-sense strand) with the

Ago-Akt mixture in a cleavage buffer (3 ug of yeast tRNA, 25 mM HEPES-KOH, pH 7.5, 50 mM

potassium acetate, 5 mM magnesium acetate, and 5 mM DTT) at 37 °C for 90 min. The reactions were

terminated by adding equal volumes of phenol:chloroform:isoamy-alcohol. RNA was precipitated with

ethanol and separated on 15% PAGE containing 7 M urea for passenger strand cleavage or on 8%

PAGE for target substrate cleavage. The specific cleavage products were indicated with an arrow based

on the ladder generated from partial digestion of input substrate by RNase T1 (T1). Cleavage efficiency

(%) was quantified by the equation: (intensity of cleavage products / intensity of total input) x 100.

For coupled in vitro kinase and target cleavage assays (as in Figure S5B) bacterially-expressed

WT or S387A Ago2 were concentrated to 3.5 – 5 mg/mL using Vivaspin 500 (30,000 MWCO) spin

columns (Sartorius). Kinase reactions were performed at 37°C for 1 hour using Ago2 and Akt3 (3:1) in 1 x

kinase buffer (Life Technologies) and 0.5 mM ATP (Life Technologies). 1 µM of KP372-1 (Echelon) or an

equivalent volume of DMSO was used to test Akt3 inhibition. In vitro cleavage reactions were performed

as above.