5884–5892 Nucleic Acids Research, 2010, Vol. 38, No. 17
`doi:10.1093/nar/gkq347
`
`Published online 10 May 2010
`
`Incorporation of pseudouridine into mRNA enhances
`translation by diminishing PKR activation
`Bart R. Anderson1, Hiromi Muramatsu2, Subba R. Nallagatla3, Philip C. Bevilacqua3,
`Lauren H. Sansing4, Drew Weissman1 and Katalin Kariko´ 2,*
`
`1Department of Medicine, 2Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA,
`3Department of Chemistry, Pennsylvania State University, University Park, PA and 4Department of Neurology,
`University of Pennsylvania, Philadelphia, PA, USA
`
`Received February 8, 2010; Revised April 8, 2010; Accepted April 20, 2010
`
`ABSTRACT
`
`Previous studies have shown that the translation
`in vitro transcribed messenger RNA
`level of
`(mRNA) is enhanced when its uridines are replaced
`with pseudouridines; however, the reason for this
`enhancement has not been identified. Here, we
`in vitro transcripts containing
`demonstrate that
`uridine activate RNA-dependent protein kinase
`(PKR), which then phosphorylates translation initi-
`ation factor 2-alpha (eIF-2a), and inhibits translation.
`In contrast, in vitro transcribed mRNAs containing
`pseudouridine activate PKR to a lesser degree, and
`translation of pseudouridine-containing mRNAs is
`not repressed. RNA pull-down assays demonstrate
`that mRNA containing uridine is bound by PKR more
`efficiently than mRNA with pseudouridine. Finally,
`the role of PKR is validated by showing that pseudo-
`uridine-
`and
`uridine-containing RNAs were
`translated equally in PKR knockout cells. These
`results indicate that the enhanced translation of
`mRNAs containing pseudouridine, compared to
`those containing uridine, is mediated by decreased
`activation of PKR.
`
`INTRODUCTION
`
`In vitro transcribed messenger RNA (mRNA) has many
`advantages as a vehicle for gene delivery. Transfection of
`mRNA is very efficient (1), and rapid expression of the
`encoded protein can be achieved. Unlike viral vectors or
`plasmid DNA, cell-delivered mRNA does not introduce
`the risk of insertional mutagenesis (2,3). Previous studies
`have shown that RNA can activate a number of innate
`immune receptors, including Toll-like receptor (TLR)3,
`TLR7, TLR8 and retinoic acid-inducible gene I (RIG-I).
`However, activation of these receptors can be avoided by
`
`incorporating modified nucleosides, e.g. pseudouridine
`( ) or 2-thiouridine (s2U), into the RNA (4,5).
`RNA-dependent protein kinase (PKR) is a ubiquitous
`mammalian enzyme with a variety of cellular functions,
`including regulation of translation during conditions of
`cell stress. During viral
`infection, PKR binds viral
`double-stranded (ds)RNA, autophosphorylates and sub-
`sequently phosphorylates the alpha subunit of translation
`initiation factor 2 (eIF-2a), thus repressing translation
`(6,7). Originally, potent activation of PKR was thought
`to require >30-bp-long dsRNA (8). It has subsequently
`been shown that PKR can be activated by a variety of
`RNA structures that include single-stranded (ss)RNA
`forming hairpins (9,10),
`imperfect dsRNA containing
`mismatches (10), short dsRNA with ss tails (11), stem–
`0
`loop structures with 5
`-triphosphates (12,13), and unique
`elements present in interferon gamma (IFN-g) and tumor
`necrosis factor-alpha mRNAs (14). Viral (15,16) and
`cellular RNAs (17–20) transcribed as ssRNA but contain-
`ing secondary structure can also be potent PKR activa-
`tors. PKR activation by short dsRNA, such as siRNA,
`has also been demonstrated (21–26). These reports
`indicate that a wide variety of RNA structures can
`activate PKR, provided they contain some dsRNA
`element. Modified nucleosides present in homopolymeric
`RNAs (27–30) or in short transcripts (25,31,32) can influ-
`ence activation of PKR. However,
`it has not been
`investigated whether modified nucleosides present
`in
`long, protein-encoding mRNAs impact activation of
`PKR.
`Previously, we demonstrated that in vitro transcribed
`mRNAs containing are translated at significantly
`higher levels than those containing unmodified uridines
`(33). However, the molecular mechanism underlying this
`enhancement has not been identified. Here, we show that
`one
`cause of
`this
`translational difference
`is
`that
` -containing mRNA activates PKR less efficiently than
`uridine-containing mRNA. This reduced PKR activation
`
`*To whom correspondence should be addressed. Tel: +1 215 662 7927; Fax: +1 215 349 8157; Email: kariko@mail.med.upenn.edu
`
`ß The Author(s) 2010. Published by Oxford University Press.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
`by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
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`also mitigates general translational inhibition of cellular
`induced when unmodified in vitro
`proteins
`that
`is
`transcribed mRNAs are delivered to cells. Since replacing
`uridines with pseudouridines also abrogates
`innate
`immune activation by RNA, -modified mRNAs are at-
`tractive vectors for gene delivery or replacement, vaccine
`antigen delivery or other RNA-based therapeutic
`applications.
`
`MATERIALS AND METHODS
`
`Cells and reagents
`
`Human embryonic kidney (HEK) 293T cells were
`obtained from the American Type Culture Collection
`and were
`cultured in Dulbecco’s modified Eagle’s
`medium (DMEM) supplemented with 2 mM L-glutamine
`(Life Technologies), 100 U/ml penicillin and 100 mg/ml
`streptomycin (Invitrogen) and 10% fetal calf serum
`(HyClone).
`Immortalized wild-type (WT) and PKR
`/–) mouse
`embryonic
`fibroblasts
`knockout
`(PKR
`(MEFs) were generously provided by Robert Silverman
`(Cleveland Clinic Foundation) and were maintained in
`RPMI medium supplemented with 2 mM L-glutamine,
`100 U/ml penicillin, 100 mg/ml streptomycin and 10%
`fetal
`calf
`serum.
`Polyinosinic:polycytidylic
`acid
`(poly(I:C)) was purchased from Sigma and polydeoxy-
`cytidylic acid (poly(dC)) was purchased from Midland
`Certified Reagent Co.
`
`mRNA synthesis
`
`RNAs were transcribed as previously described (4), using
`linearized plasmids encoding firefly luciferase (pT7TS-
`fLuc and pTEVluc) or Renilla luciferase (pT7TS-Ren)
`and T7 RNA polymerase (Megascript, Ambion). Except
`where otherwise specified, capped mRNA was generated
`by performing transcription in the presence of cap analog
`0
`0
`0
`-O-Me-m7G(5
`)ppp(5
`)G (New England Biolabs). All
`3
`0
`mRNAs were transcribed to contain 30 or 50-nt-long 3
`poly(A) tails. Triphosphate-derivatives of , s2U, m5C,
`m6A and m5U (TriLink) were used in place of their
`cognate
`unmodified NTP
`to
`generate modified
`nucleoside-containing RNA. Following transcription, the
`template plasmids were digested with Turbo DNase and
`RNAs were precipitated with 2.5 M lithium chloride at
`20
`
`C for 4 h. RNAs were pelleted by centrifugation,
`washed with 75% ethanol and then reconstituted in
`nuclease-free water. The concentration of RNA was
`determined by measuring the optical density at 260 nm.
`All RNA samples were analyzed by denaturing agarose
`gel electrophoresis for quality assurance. Each RNA
`type was synthesized in 4–10 independently performed
`transcription experiments and all experiments were per-
`formed with at least two different batches of mRNA.
`Enzymatic capping was performed using ScriptCap m7G
`capping kit
`(Epicentre) on mRNA transcribed with
`0
`-[g-32P]-triphosphate
`(GE Healthcare).
`guanosine
`5
`Efficiency of capping was verified by monitoring the elim-
`ination of g-32P from the mRNA. Biotinylated mRNA
`was transcribed with the addition of 1:5 biotinylated
`
`Nucleic Acids Research, 2010, Vol. 38, No. 17 5885
`
`CTP (Roche Applied Sciences)
`reaction.
`
`in the transcription
`
`Detection of reporter proteins in RNA-transfected cells
`Cells were seeded into 96-well plates at a density of
`5.0  104 cells/well 1 day prior to transfection. RNA was
`complexed with lipofectin (Invitrogen) as described previ-
`ously (4). Cells were exposed to 50 ml DMEM containing
`lipofectin-complexed RNA (0.25 mg) for 1 h, which was
`then replaced with complete medium and further
`cultured. Cells were lysed in 25 ml firefly, Renilla, or
`dual-luciferase specific lysis reagents (Promega). Aliquots
`of 2 ml were assayed with the corresponding enzyme sub-
`strates and a LUMAT LB 950 luminometer (Berthold) at
`a 10-s measuring time.
`
`Assessment of total protein synthesis
`HEK293T cells were seeded into 96-well plates at a density
`of 5.0  104 cells/well with 1000 U/ml
`interferon-aA/D
`(Sigma) 1 day prior to transfection. Cells were incubated
`in methionine/cysteine-free medium (Invitrogen) for 1 h,
`then pulsed with complete medium supplemented with
`35S-methionine/cysteine (140 mCi/ml) (PerkinElmer) for
`1–3 h. Cells were lysed in RIPA lysis buffer supplemented
`with protease inhibitor cocktail
`(Sigma). Lysate was
`diluted in 0.1% bovine serum albumin (BSA), and macro-
`molecules were precipitated by the addition of trichloro-
`acetic acid (TCA) and 30 min incubation on ice.
`Precipitates were filtered onto glass microfiber filters
`(Whatman) and washed with 10% TCA and 100%
`35S-methionine/cysteine was
`ethanol.
`Incorporated
`quantified using Ecolite(+) scintillation cocktail
`(MP
`Biomedicals) and a Beckman LS 6000IC scintillation
`counter.
`
`PKR activation in vitro
`
`(11) was
`described
`as
`PKR prepared
`Purified
`dephosphorylated using lambda protein phosphatase
`(New England Biolabs). Final concentrations of 0.75 mM
`dephosphorylated PKR, 0.1 mM ATP and 0.15 mCi/ml ad-
`0
`-[g-32P]-triphosphate (g-32P-ATP) (PerkinElmer)
`enosine 5
`were mixed with the indicated concentration of RNA for
`
`10 min at 30
`C in a buffer consisting of 4 mM MgCl2,
`100 mM KCl and 20 mM HEPES, pH 7.5. The reaction
`was stopped by the addition of NuPage LDS sample
`buffer and reducing agent (Invitrogen) and heating for
`
`C. Unincorporated g-32P-ATP was separated
`10 min at 70
`from radiolabeled PKR by running samples on a 12%
`sodium dodecyl sulfate polyacrylamide gel electrophoresis
`(SDS–PAGE) gel. Phosphorylated PKR was imaged in
`dried gels using a phosphor storage screen (Molecular
`Dynamics) and detected using Storm or Typhoon
`Phosphorimagers (GE Healthcare). Band densities were
`quantified using ImageQuant software (GE Healthcare).
`
`Western blotting
`HEK293T cells were seeded into 96-well plates at a density
`of 5.0  104 cells/well, with 1000 U/ml interferon-aA/D 1
`day prior to transfection. At the indicated time following
`
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`5886 Nucleic Acids Research, 2010, Vol. 38, No. 17
`
`RNA transfection, cells were lysed in RIPA lysis buffer
`supplemented with protease
`inhibitor
`cocktail and
`HALT phosphatase inhibitor (Pierce). Equal mass of
`protein (1030 mg per sample) was loaded onto a 12%
`SDS–PAGE gel. Proteins were subsequently transferred
`to
`a Hybond-P polyvinylidene
`fluoride
`(PVDF)
`membrane (GE Amersham), blocked with 2.5% non-fat
`milk in TBS containing 0.05% Tween-20, and probed with
`antibodies
`for PKR-pT446 and PKR (Epitomics),
`eIF-2a-pS51 and eIF-2a (Cell Signaling Technologies),
`or PABP (Abcam). Membranes were stripped by agitating
`gently in a buffer of 2% SDS, 100 mM b-mercaptoethanol,
`
`C, then subsequent-
`62.5 mM Tris pH 6.7 for 30 min at 50
`ly re-blocked and re-probed. Image was captured using the
`Fujifilm LAS1000 digital imaging system. Linear bright-
`ness and contrast were adjusted using GIMP 2.6 software.
`
`Biotinylated RNA pull-down
`
`HEK293T cells were lysed in RIPA lysis buffer supple-
`mented with protease inhibitor cocktail and RNase inhibi-
`tor (RNasin, Promega). Biotinylated mRNA (2 mg) was
`added to 25 ml
`lysate and incubated on ice for 2 h.
`Subsequently, 50 ml of streptavidin-agarose bead 50%
`slurry (Invitrogen) was added and incubated on ice for
`1 h. Beads with bound RNA and proteins were centrifuged
`and washed, and proteins were released from RNA by
`
`C for 10 min in the presence of
`heating samples at 70
`NuPage LDS sample buffer and reducing agent. Samples
`were separated by 10% SDS–PAGE and transferred to
`PVDF membranes. PKR and poly(A)-binding protein
`(PABP) were detected by western blotting.
`
`Statistical analysis
`
`All data are reported as mean ± standard error of the
`mean (SEM). Statistical differences between treatment
`groups were calculated by the Student’s t-test using
`Microsoft Excel. For all statistical testing, a P-value
`<0.05 was considered significant.
`
`RESULTS
`Conventional in vitro transcribed mRNA induces
`translational repression
`
`We previously reported that mRNA transcribed in vitro
`containing in place of uridine is translated more effi-
`ciently than mRNA containing unmodified nucleosides
`(33). In order to determine whether the translational en-
`hancement exerted by incorporated into RNA is re-
`stricted to the modified transcript or also extends to
`unmodified transcripts, we performed co-transfection ex-
`periments delivering equal amounts of Renilla and firefly
`luciferase-encoding mRNAs to cells. As expected, the
`mRNAs were translated much more efficiently when
`both contained as compared to when both were un-
`modified (Figure 1A). However, when only one of the
`mRNAs contained modification, the translation level
`of the -containing RNA decreased (50%) relative to
`the level measured when both contained . One explan-
`ation for these findings could be that unmodified RNA
`
`inhibition by unmodified in vitro transcribed
`Figure 1. Translational
`mRNA. (A) In vitro transcribed mRNAs encoding Renilla luciferase
`(Ren) and firefly luciferase (Luc) were synthesized with and without
` modifications then mixed (1 : 1 mass ratio) as indicated. The mixed
`mRNA was complexed with lipofectin and added to HEK293T cells
`seeded in 96-well plates (0.25 mg RNA/well). Cells were lysed 4 h after
`transfection and dual
`luciferase measurements were performed in
`aliquots (1/20th) of the lysates. Values presented are normalized to
`cells transfected with Ren and Luc mRNAs when both contained
`modifications. Error bars indicate the standard error of n = 3 samples.
`(B) Unmodified or pseudouridine-containing RNA was delivered to
`HEK293T cells by lipofection. Cells were subsequently incubated
`with 35S-methionine/cysteine
`supplemented medium,
`lysed,
`and
`proteins were TCA precipitated. Data are presented as percentage of
`counts obtained from mock transfected cells. Data shown are mean
`values from three independent experiments ± SEM.
`
`inhibits the translation of the co-delivered RNA, while
` -containing RNA has no such inhibitory effect. To
`explore whether
`translation of
`endogenous
`cellular
`mRNAs are similarly influenced by exogenously delivered
`in vitro transcribed mRNAs, total cellular protein synthe-
`sis was monitored in cells transfected with mRNA con-
`taining modification or no modification. Both types of
`mRNA reduced cellular protein translation; however, the
`suppression of protein synthesis was greater with unmodi-
`fied RNA than with -containing RNA (Figure 1B).
`PKR-activating poly(I:C) and non-activating poly(dC)
`were used as controls. Mock transfected cells were
`treated with the transfection reagent (lipofectin) only,
`without nucleic acid.
`
`Conventional in vitro transcribed mRNA activates PKR
`
`To determine whether the inhibition of translation by un-
`modified mRNA is mediated by PKR, in vitro transcribed
`
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`mRNAs were first analyzed in a cell-free system using
`purified PKR. Four different mRNAs were tested: un-
`modified and -modified mRNA, each with either a cap
`0
`0
`In vitro
`-end (5
`ppp).
`or a triphosphate at
`their 5
`0
`ppp and containing uridines
`transcribed mRNA with 5
`activated PKR to a greater extent than those containing
` (Figure 2). This
`reduced activation of PKR by
` -containing transcripts is consistent with the previously
`in vitro translation from
`observed enhancement of
` -containing RNA in rabbit reticulocyte lysates (33).
`0
`ppp on short RNAs has previously
`Since the presence of 5
`been shown to enhance the activation of PKR (12,13), it
`0
`ppp present on
`was important to determine whether the 5
`long mRNAs also contributed to PKR activation. To
`0
`in
`vitro
`ppp,
`transcripts were
`capped
`remove
`5
`enzymatically (Supplementary Figure S1), which com-
`0
`ppp, and then tested. As Figure 2
`pletely removed the 5
`0
`ppp on un-
`demonstrates, the presence or absence of 5
`modified and -modified transcripts did not significantly
`alter their ability to activate PKR. It has been shown that
`a variety of nucleoside modifications in RNA can influ-
`ence the activation of RNA sensors (4,5,32); therefore, the
`effect of incorporating the modified nucleosides s2U,
`5-methylcytidine (m5C), 6-methyladenosine (m6A) or
`5-methyluridine (m5U) into mRNA was also analyzed.
`mRNA containing s2U, m5C or m6A activated PKR to
`a lesser extent than unmodified RNA, while RNA with
`m5U activated
`PKR to
`the
`greatest
`extent
`(Supplementary Figure S2).
`
`Pseudouridine-containing mRNA does not
`activate PKR in cells
`Next, we investigated the impact of -containing mRNA
`on PKR activation in the complex cellular environment.
`
`Nucleic Acids Research, 2010, Vol. 38, No. 17 5887
`
`Following control studies demonstrating that RNAs with
`or without nucleoside modification can be delivered to
`cells with the same efficiency (data not shown), unmodified
`or -containing mRNA was complexed with lipofectin
`and delivered into HEK293T cells. PKR activation was
`assessed by western blot using an antibody specific for
`PKR phosphorylated on Thr446, a site at which phos-
`phorylation is
`requisite
`for PKR activation (34).
`Consistent with the results observed using purified PKR,
`transfection of unmodified transcript induced PKR phos-
`phorylation, which was dramatically reduced if the trans-
`fected RNA contained (Figure 3A). Similarly,
`incorporation of s2U or m5C into RNA reduced the
`level of PKR phosphorylation relative to that induced
`by unmodified RNA, while m5U incorporation into
`RNA enhanced PKR phosphorylation (Supplementary
`Figure S3A). Incorporation of m6A into RNA also
`enhanced PKR phosphorylation
`in
`cells,
`despite
`reducing PKR activation in vitro.
`Phosphorylation of eIF-2a, a substrate of PKR, was
`induced in HEK293T cells by transfection with unmodi-
`fied RNA but not with -containing RNA (Figure 3B).
`Incorporation of modified nucleosides other than into
`mRNA altered the phosphorylation of eIF-2a in direct
`parallel
`to their alterations of PKR phosphorylation
`(Supplementary Figure S3B).
`
`Translation of unmodified mRNA is enhanced upon
`inhibiting or eliminating PKR
`
`Viral proteins C8L of swinepox and K3L of vaccinia are
`inhibitors of PKR and have been shown to reverse
`PKR-mediated inhibition of translation in mammalian
`cells (35). Thus, to confirm the role of PKR in the trans-
`lational differences observed between uridine- and
`
`Figure 2. Activation of purified PKR by in vitro transcribed RNA. Purified PKR was incubated with g-32P-ATP and in vitro transcribed mRNA for
`10 min. Reaction products were separated by SDS–PAGE and imaged using phosphor storage radiography. Unmodified or -containing mRNAs
`0
`-ends. Complete capping of RNA was achieved post-transcriptionally using
`encoding firefly luciferase contained triphosphates (ppp) or cap at their 5
`vaccinia capping enzyme. Concentration of mRNA in reactions was 3.1, 6.2, 12.5 and 25 mg/ml. Quantified phosphorylation is presented as a bar
`graph below each band. Values were normalized to those obtained with 25 mg/ml uncapped, unmodified RNA. No RNA () and 79 bp dsRNA were
`used as negative and positive controls.
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`5888 Nucleic Acids Research, 2010, Vol. 38, No. 17
`
`Figure 3. PKR activation by in vitro transcribed mRNA in cells.
`Unmodified or -containing in vitro transcribed firefly luciferase
`mRNA was delivered to cells by lipofection. Following RNA transfec-
`tion, cells were lysed at 4 h (A) or at the indicated time (B), proteins
`were separated by SDS–PAGE, and assayed for phosphorylation of
`PKR (A) or eIF-2a (B) by western blotting. No RNA (), poly(dC)
`and poly(I:C) were used as controls. Relative phosphorylation is
`indicated below each gel
`lane, calculated as phosphorylated band
`density divided by total band density and then normalized to the phos-
`phorylation induced by unmodified RNA.
`
` -containing transcripts, we utilized C8L, K3L and two
`K3L mutants: hyperactive K3L-H47R and inactive
`K3L-Y76A (35,36). Based on the premise that PKR is
`activated by in vitro transcribed mRNAs that contain
`uridine but not by those with ,
`inhibition of PKR
`would be expected to increase the translation of unmodi-
`fied mRNA but to have no effect on the translation of
` -containing RNA. Indeed, in the presence of PKR in-
`hibitors, the amount of translation increased from un-
`modified transcripts but not from -modified transcripts
`(Figure 4A).
`Further evidence confirming the role of PKR in sup-
`pressing translation of unmodified mRNAs was obtaining
`using mouse embryonic fibroblasts (MEFs) derived from
`PKR-knockout animals. In wild-type MEFs, translation
`of -containing transcripts was 4–5-fold greater than that
`of unmodified transcripts (Figure 4B). In PKR-deficient
`MEFs, however, the extent of translation of -modified
`mRNA was not different from that of unmodified mRNA.
`Additionally, RNA transfection does not induce phos-
`phorylation of eIF-2a in PKR-deficient MEFs, as it does
`in WT cells (Figure 4C). These results demonstrate that
`the activity of PKR is necessary for the decreased trans-
`lation of unmodified transcripts relative to -containing
`transcripts.
`
`Pseudouridine-containing mRNA is not bound by PKR
`To test whether -modified mRNA is a competitive in-
`hibitor of PKR, a 200-bp dsRNA known to activate PKR
`was mixed with a 5–125-fold mass excess of -modified
`
`RNA. All concentrations of -modified RNA tested failed
`to inhibit the activation of PKR by the 200-bp dsRNA
`(Figure 5). Similarly, a 125-fold mass excess of mRNA
`containing s2U, m5C or m6A did not inhibit PKR activa-
`tion by dsRNA (Figure S4). The results were the same
`using lower mass excess, equal mass or equal molar
`mixes (data not shown), demonstrating that RNAs con-
`taining modified nucleosides are not competitive inhibitors
`of PKR. The lack of PKR inhibition by transcripts con-
`taining modified nucleosides suggests a lack of binding
`between PKR and modified RNAs. To directly test
`binding, biotinylated transcripts having
`30-nt-long
`poly(A) tails and containing either or uridines were
`mixed with HEK293T cell lysates, and complexes were
`then precipitated using
`streptavidin-agarose beads.
`Western blots of the precipitates indicated that PKR
`bound to unmodified RNA, but bound poorly to
` -modified RNA (Figure 6), consistent with reduced ac-
`tivation of PKR by -containing RNA. By contrast,
`poly(A)-binding protein (PABP) bound equally well to
`both transcripts. These results indicate that unmodified
`RNA, but not -modified RNA, binds to and activates
`PKR.
`
`DISCUSSION
`
`We demonstrate that modified nucleosides in mRNA
`reduce PKR activation and identify a mechanism by
`which -incorporation in mRNA enhances translation
`of the encoded protein. Our data show that conventional
`in vitro transcribed RNA inhibits translation of reporter
`and cellular mRNAs, in part through the activation of
`PKR. However, this inhibitory activity is not induced by
` -containing mRNA. Using multiple lines of investiga-
`tion, our studies demonstrate that unmodified in vitro
`transcribed mRNA activates PKR, resulting in phosphor-
`eIF-2a and inhibition of
`ylation of
`translation.
`0
`0
`ppp with 5
`cap structure on the
`Replacement of 5
`mRNA does not substantially alter this PKR activation.
`Examining translation in the context of PKR inhibitors
`and in PKR-deficient cells confirmed that enhanced trans-
`lation of -containing mRNA is a consequence of dimin-
`ished
`PKR activation. Mechanistically, modified
`nucleoside incorporation reduces RNA recognition by
`PKR. This is supported by data demonstrating that
`RNAs containing modified nucleosides do not inhibit
`PKR activation by dsRNA and that PKR binds poorly
`to -containing RNA.
`PKR activation by unmodified RNA has a more
`pronounced impact on translation of
`the transfected
`reporter mRNA than on total
`cellular
`translation
`(Figure 1). A similar local translation effect has been
`observed with PKR activation by IFN-g mRNA (19,37).
`The pronounced local
`inhibition is likely due to the
`kinetics of phosphorylation and dephosphorylation of
`PKR. Activated PKR most dramatically inhibits local
`translation because rapid dephosphorylation of PKR
`limits the impact on more distant translation. Therefore,
`translation of a PKR-activating mRNA is more severely
`impacted than total cellular translation. Furthermore, the
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`
`Figure 4. Translation of in vitro transcribed mRNA in the absence of PKR activity. (A) HEK293T cells were transfected with plasmids encoding
`protein inhibitors of PKR: swinepox C8L protein, wt vaccinia K3L, hyperactive K3L-H47R, inactive K3L-Y76A, or pG5 empty vector. Twenty-four
`hours later, unmodified or -modified in vitro transcribed mRNAs encoding firefly luciferase were delivered by lipofection, and luciferase activity was
`measured 4 h later. Data were normalized to values obtained when cells were first transfected with empty vector then with unmodified RNA.
`Presented data are mean values from three replicates ± SEM. (B) MEF cell lines derived from wild-type (WT) or transgenic mice that do not
`/
`) were transfected with unmodified or -containing in vitro transcribed mRNAs encoding firefly luciferase. Data
`express functional PKR (PKR
`were normalized to values obtained when cells were transfected with unmodified RNA and expressed as fold increase in translation of -containing
`mRNA over unmodified RNA. Values are from three replicate wells ± SEM, and are representative of at least three independently performed
`/– MEF cells were transfected with unmodified or -containing in vitro transcribed mRNAs encoding firefly
`experiments. (C) WT and PKR
`luciferase, or mock transfected with no RNA (–). Cells were lysed 2 h following RNA transfection; proteins were then separated by SDS–PAGE
`and assayed for eIF-2a phosphorylation by western blotting. Relative phosphorylation is indicated above each gel lane, calculated as phosphorylated
`band density divided by total band density and then normalized to the phosphorylation induced by unmodified RNA in wild-type cells. Absence of
`PKR was also confirmed by western blotting.
`
`Figure 5. -containing mRNA does not inhibit PKR activation. An
`activating 200 bp dsRNA was mixed with a 5–125-fold mass excess
`of -containing in vitro transcribed firefly luciferase mRNA prior to
`incubation with purified PKR. Reaction products were separated by
`SDS–PAGE. Relative band densities are presented below each gel
`lane and normalized to dsRNA only. Data shown are representative
`of three independent experiments.
`
`observation that -containing RNA also causes some re-
`duction in total protein synthesis suggests that there are
`additional effects on cellular translation which are not
`mediated by PKR.
` -containing RNA activates PKR more effectively
`in vitro as compared to in vivo (Figures 2 and 3). One
`possible reason for this difference is that PKR activation
`in vivo occurs in the presence of competing factors such as
`phosphatases, components of the translational system and
`other proteins affecting the structure and accessibility of
`the RNA to PKR. In contrast, in vitro assays lack such
`competing factors that would limit or reverse PKR
`phosphorylation.
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`
`Figure 6. -containing mRNA does not pull-down PKR. Biotinylated
`in vitro transcribed unmodified or -containing RNAs were incubated
`with HEK293T cell lysates for 2 h. The RNA and bound proteins were
`pulled down using streptavidin-agarose beads. An aliquot of lysate that
`was incubated only with beads but without RNA () was also pro-
`cessed. Aliquots of pull-down proteins as well as the supernatants were
`separated by SDS–PAGE. PKR and PABP were detected by western
`blotting. Relative band densities of PKR divided by PABP compared to
`unmodified RNA are presented below each gel lane.
`
`Although mRNA is normally transcribed without a
`complementary antisense transcript or long stretches
`of
`self-complementarity,
`it contains many short ds
`regions and other intramolecular secondary structures
`(Figure S5). In addition to long perfectly dsRNA, PKR
`is activated by RNA that contains either hairpins (9),
`bulges, mismatched base-pairing (10),
`short
`internal
`dsRNA regions
`(11) or unique structures naturally
`present in selected cellular mRNAs (17–20). As previously
`demonstrated for TLR3 (38), it is likely that the activation
`of PKR by in vitro transcribed mRNA is due to the for-
`mation of intra- and intermolecular secondary structures.
`PKR is then activated upon binding to these structures,
`similar to the classical dsRNA-mediated mechanism of
`PKR activation. Nucleoside modifications influence base
`pairing and secondary structure formation (39–46), which
`likely contribute to their effects on PKR activation.
`Alterations to the shape of the helix formed and interrup-
`tions to the minor groove, which is presumed to be the
`principal
`location of PKR interaction with RNA
`(32,47,48), are also likely to play significant roles in
`determining how each modified nucleoside will
`impact
`RNA-mediated PKR activation.
`Unlike short ssRNAs (12), PKR activation by long
`in vitro transcribed mRNA is not dependent on the
`0
`-triphosphate, as mRNA containing
`presence of a 5
`0
`ppp with cap structure also ac-
`complete replacement of 5
`tivates PKR (Figures 2 and S1). The difference between
`0
`ppp in the
`these findings might reflect the amount of 5
`RNAs being compared. Forty-seven nucleotide-long
`ssRNA induced 100-fold more PKR activation when the
`0
`-end contained triphosphates (12), while our data did
`5
`0
`not show any significant effect of removing the 5
`ppp
`from 1976-nt-long mRNA, which contains 40-fold less
`0
`ppp. Our finding is more consistent with the result
`5
`reported for 47-bp-long dsRNA wherein PKR activation
`0
`ppp (12).
`did not depend on 5
`Previous reports indicate that PKR activation is altered
`by the presence of modified nucleosides in homopolymeric
`RNA (27,28,30) and short ssRNA and dsRNA (32). Our
`data extend these findings by demonstrating that
`
`incorporation of modified nucleosides into long in vitro
`transcribed mRNA also alters activation of PKR, and
`subsequent translation of the RNA. We observe substan-
`tial PKR activation by in vitro transcribed mRNA, which
`is reduced by incorporation of . Additionally, our
`studies show reduced PKR activation by mRNA that
`contains m5C, enhanced PKR activation by mRNA con-
`taining m5U and elimination of PKR activation by
`s2U-containing mRNAs. These results vary from those
`obtained when testing PKR activation by short 47 nt
`ssRNA: a low level of PKR activation by unmodified
`RNA, which was dependent on the presence of a
`0
`5
`-triphosphate, and near-complete elimination of PKR
`activation by incorporation of modified nucleosides (32).
`However, when testing short 47-bp dsRNA, the effects
`observed were similar to those reported here: PKR acti-
`vation by unmodified RNA, which is reduced by incorp-
`oration, increased by m5U incorporation, and eliminated
`by s2U incorporation. This similarity to short dsRNA,
`and dissimilarity to ssRNA, supports our model that
`PKR activation by long in vitro transcribed mRNA is
`due to regions of secondary structure formed within the
`0
`RNA and is independent of the 5
`-end.
`Unlike the other nucleoside modifications tested, the
`presence of m6A in mRNA impacted PKR activation dif-
`ferently in vivo than in vitro. In vitro, mRNA containing
`m6A activated PKR only moderately (Figure S2) whereas
`in vivo, m6A-containing mRNA activated PKR more
`potently than unmodified RNA (Figure S3). Although
`the significance of this observation is not fully understood,
`the discrepancy may be explained by the presence of add-
`itional factors in cells that facilitate increased ds formation
`in m6A-containing mRNA in vivo.
`Nucleic acids containing modified nucleosides can act as
`antagonists of nucleic acid-sensing TLRs
`(49–52).
`Therefore, we
`asked whether mRNAs
`containing
`modified nucleosides inhibit activation of PKR by its
`cognate ligand, dsRNA. PKR is
`still activated by
`dsRNA in the presence of a 125-fold excess of mRNA
`containing or other modified nucleosides (s2U, m5C
`or m6A), indicating that mRNAs containing modified nu-
`cleosides are not inhibitors of PKR (Figures 5 and S4).
`This extends previous data demonstrating that short
`ssRNAs containing modified nucleosides do not inhibit
`PKR (32). Furthermore, in cell lysates, RNA containing
` pulls down less PKR than RNA containing uridine
`(Figure 6). This reduction in PKR binding is consistent
`with prior in vitro data demonstrating small reductions in
`PKR binding to short dsRNA and ssRNA that contain
`modified nucleosides (32). From t