ires mediated translational regulation of p53 isoforms

Advanced Review

IRES mediated translationalregulation of p53 isoformsArandkar Sharathchandra, Aanchal Katoch and Saumitra Das∗

p53 is a well known tumor suppressor protein that plays a critical role incell cycle arrest and apoptosis. It has several isoforms which are produced bytranscriptional and posttranscriptional regulatory mechanisms. p53 mRNA hasbeen demonstrated to be translated into two isoforms, full-length p53 (FL-p53)and a truncated isoform �N-p53 by the use of alternative translation initiationsites. The mechanism of translation regulation of these two isoforms was furtherelucidated by the discovery of IRES elements in the p53 mRNA. These two IRESswere shown to regulate the translation of p53 and �N-p53 in a distinct cell-cyclephase-dependent manner. This review focuses on the current understanding ofthe regulation of p53 IRES mediated translation and the role of cis and trans actingfactors that influence expression of p53 isoforms. © 2013 John Wiley & Sons, Ltd.

How to cite this article:WIREs RNA 2014, 5:131–139. doi: 10.1002/wrna.1202

INTRODUCTION

p53 is a tumor suppressor protein that plays akey role in maintaining genome integrity and

preventing cellular transformation.1 Approximately50% of human tumors have been seen to havemutations in this gene.2 Its involvement in cell cycle,apoptosis, stem cell differentiation , senescence, andDNA repair1,3,4 exemplifies its role as a masterregulator. Under stress conditions such as DNAdamage, replication stress or telomere dysfunction,the ATM/ATR-Chk1/2 kinase cascade upregulatesp53 while under oncogenic stress the alternativeARF pathway is involved in its activation. p53 isa transcription factor that binds to target DNAsequences via its transactivation domains and controlsthe expression of protein coding genes as wellas micro-RNAs (miRNAs).These miRNAs in turnregulate several different cellular mRNAs involvedin key cellular functions such as apoptosis, cell cyclearrest, migration, epithelial–mesenchymal transition,stemness, and metabolism.5

∗Correspondence to: [email protected]

Department of Microbiology and Cell Biology, Indian Institute ofScience, Bangalore, India

Conflict of interest: The authors have declared no conflicts ofinterest for this article.

p53 protein has several isoforms, which aregenerated by alternate splicing, internal promoter andinternal initiation. Carboxyl terminal of p53 variantsare called TA isoforms and amino terminal variantsare � (delta) isoforms. It has been shown that p53gene has two internal promoters; this suggests thatp53 gene has dual gene structure like its ancestral pro-teins p63 and p73. Dual gene structure of p53 is alsoconserved across the species, including Drosophilaand Zebrafish. p53 mRNA intron-9 and intron-2are spliced in different ways resulting in different Nterminal and C terminal variants. Altogether humanp53 gene can express 12–13 different isoforms ofthe protein (full-length p53 or FLp53 or TAp53α,TAp53β, TAp53γ , �40p53α, �40p53β, �40p53γ ,�133p53α, �133p53β, �133p53γ , �160p53α,�160p53β, �160p53γ ; also p53 AD containinga severely truncated DNA-binding domain). Theseare generated by alternative splicing, alternativepromoter usage and internal initiation of translationmechanism.6

p53 isoform lacking first 40 amino acids is called�40p53 or �N-p53 or p53/47. FL-p53 is producedfrom first AUG (present in exon 2) of p53 mRNA,whereas �N-p53 isoform is produced from an in-frame second AUG present at 252–254 nuceotides ofp53 mRNA (present in exon 4) and this isoform of p53lacks transactivation domain-I (TAD-I).7 The human�N-p53 and its mouse counterpart p44 also lacks

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the first 39 and 40 amino acids (TAD-I) respectively.This domain, apart from transactivating p53 targetgenes also interacts with MDM2 protein and targetsp53 for proteosomal degradation.8 Two independentstudies showed that �N-p53 is synthesized from sec-ond AUG by internal initiation of translation, butit is not because of either calpain cleavage or auto-proteolytic cleavage of p53 molecule.7,9 Presently it iswell established that synthesis of �N-p53 from p53mRNA occurs by Internal Ribosome Entry Site (IRES)mediated translation. Initially it was thought that �N-p53 oligomerizes with p53 and negatively regulatesthe transcription and growth suppressive functions.7

Increasing evidences suggest that �N-p53 has uniquetranscriptional targets and functions.10 �N-p53 eitheralone or in combination with p53 is able to induceapoptosis.9,11 Incidentally, the mouse homolog of �N-p53, the �40p53 is also deficient in the transactivationdomain and is highly expressed in mouse embryonicstem cells (ESC).12 Over expression of shorter isoformof p53 in p44 transgenic mice causes early ageing, thisstudy reveals that expression of p53 and �N-p53 isrequired in appropriate levels to maintain a balancebetween tissue regeneration and tumor suppression.13

It was observed that even in the absence of firsttransactivation domain, ‘truncated’ p53 induces manyapoptosis related genes which were unique and arenot induced by p53.14Also it has been reported that�N-p53 regulates folding, oligomerization, and post-translational modification of p53 complexes, thereforeeffecting a change in the gene expression.10 �40p53,prolongs pluripotency and inhibits the progression todifferentiated form in embryonic stem cells and actsas a switch from pluripotent ESC to differentiatedsomatic cells.12 Over expression of �40p53 decreasespancreatic β cell proliferation, regulates glucose home-ostasis and causes early ageing in mice.15 Recentlyit has been reported that samples from mucinoushuman ovarian cancer patients showed elevated �N-p53 expression when compared to normal ovariantissues; also high �N-p53 expression was an indepen-dent prognostic marker for recurrence-free but notfor overall survival in patients suffering with ovariancancer.16 Profiling of �N-p53 and p53 in glioblas-toma revealed presence of both the isoforms, neitherof which were present in nontumor brain tissue; also itwas observed that �N-p53 was uniquely expressed ingliosis and neuronal progenitor cells.17 These studieshighlight the importance of differential expression ofp53 isoforms, however it is still unclear as to howthese two p53 isoforms are regulated under differentcellular stresses and physiological conditions. The cur-rent review focuses on translational regulation of p53isoforms by IRES mediated translation.

IRES MEDIATED TRANSLATION OFCELLULAR mRNAA cap independent recruitment of ribosome was ini-tially discovered in naturally uncapped PicornavirusmRNAs.18,19 Picornavirus mRNAs translation ini-tiates in a cap independent manner by recruitingribosome directly on to structured RNA region. Thesestructured regions were initially known as ribosomelanding pads, later renamed as Internal RibosomeEntry Site (IRES). Later observations providedevidence that some of Picornaviral proteases cleaveeIF4G to N and C terminal halves thereby interferingwith cap dependent mRNA translation.20–23 EMCVand Poliovirus infections lead to dephosphorylationof eIF4E-BP which binds to eIF4E and inhibitsthe formation of eIF4F initiation complex therebyinterfering with global cap dependent translation.24

After the discovery of IRESs in viral mRNA, itwas also shown that cellular mRNA which encodesfor immunoglobulin heavy chain binding protein(BiP) contains IRES element.25 Later observationsrevealed that several cellular mRNAs consists IRESelements in 5′UTR region, which play a majorrole in different cellular functions like cell growth,development, differentiation, proliferation, senes-cence and apoptosis.20,26 Under many cellular stressconditions cap dependent translation decreases due tomodification of cap dependent translation machinerylike cleavage of different cellular initiation factorsby viral proteases and cellular caspase proteins.27 Insuch conditions IRES mediated translation is active incell. Around 10% of cellular mRNA are estimated tohave IRES.28 Many stresses like nutrient deprivation,DNA damage and hypoxia promotes IRES mediatedtranslation of cellular mRNAs encoding some ofthe growth factors, transcription and translationfactors. Two mRNAs encoding for the cat-1Arg/Lystransporter (CAT-1) and the sodium-coupled neutralamino acid transporter (SNAT2) are translationallyregulated during amino acid starvation. CAT-15′UTR allows transient structural remodeling of itsRNA to form an active IRES, which is essential forthe translation of CAT-1 protein under amino acidstarvation conditions in mammalian cells. Undersimilar stress conditions SNAT-2 protein levels alsoget induced through IRES mediated translation.29–32

BIP and BAG-1 mRNAs also contain IRES in their5′UTR, which gets activated during heat shock andpromotes more synthesis of the respective proteins.33

IRES mediated translation also plays a major role onDNA damage. Some antiapoptotic proteins like BCL2and XIAP2 contains IRES in their 5′UTR of mRNAs,which allows their translation, when cap-dependenttranslation is inhibited.34,35 During DNA damage

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WIREs RNA Regulation of p53 translation

and cell division, serine hydroxymethyltransferase 1(SHMT1) is involved in the thymidylate biosynthesispathway. SHMT1 is known to have an IRES in its5′UTR, which allows synthesis of the protein on UVtreatment.36Another stress where IRES plays it role ishypoxia. Vascular endothelial growth factor (VEGF),involved in the development of increased vascula-ture, possesses two IRESs that enable the proteinexpression even in hypoxic cells, where translation isrepressed. Similarly IRESs present in FGF2 and PDGFare also important for cell survival under hypoxicconditions.37Activity of these cellular IRESs is criticalfor cell survival and fate. However, there are fewcellular mRNAs whose translation can be initiatedboth in cap dependent and IRES dependent manner.For example, aneurogranin mRNA which encodes forneuronal calmodulin binding protein, can be trans-lated by both the mechanisms.38Mutations in IRESsalso affects the IRES efficiency. It has been reportedin some diseases. For example C to U mutation inthe c-myc IRES causes enhanced c-myc translationand that is responsible for multiple myelomas.39

However under various cellular stress conditionsdifferent cellular IRESs respond differently. In case ofbreast cancer cells IRES mediated translation of p53is inactivated due to reduced levels of Arl2 (ADP ribo-sylation factor like 2), which leads to upregulation ofRNA pol-1 activity and hence alterations in ribosomebiogenesis.40

But how these IRES elements recruit theribosome is not clearly known. Most of the cel-lular IRESs are rich in GC content and predictedto be highly structured. It has been shown thatweaker the secondary structure of cellular IRESs,stronger is the activity.41So far no two commoncellular IRESs have shown conserved secondarystructure.26

Strength of the IRES activity is majorlyinfluenced by the RNA–protein interactions, whichinfluence the ribosome recruitment on to IRESand translation initiation. IRES elements havebeen demonstrated to utilize cellular proteins asnon-canonical initiation factors. The function of IRESelements, thus, depends on coordinated interplaybetween important cis acting elements present withinmRNA and cellular proteins functioning in trans ascritical trans-acting factors.42

TRANSLATION REGULATION OF P53mRNA

p53 might be translationally regulated was firstobserved by treating ML-1 cells with proteinsynthesis inhibitor cycloheximide that prevented

the increase in p53 levels under ionizing radiation(IR).43 Metabolic labeling with 35S-methionine andsubsequent immunoprecipitation of the p53 proteinalso showed that newly synthesized p53 accumulatesunder IR treatment. Additionally it was noticed thatp53 mRNA associates with heavier polysomes inthe cell following DNA damage, these observationsfurther confirmed the p53 regulation at translationlevel.44,45

IDENTIFICATION OF IRES ELEMENTSIN p53 mRNA

Initially it was discovered that full length p53(FL-p53) and its N-terminal truncated isoform(�N-p53) are produced by the internal initiationmechanism or alternate translation mechanism.7,9

Later it was reported that p53 mRNA has twoInternal Ribosome Entry Site (IRES) elements, FL-p53 and �N-p53 are produced by IRES mediatedtranslation from p53 mRNA. FL-p53 is producedfrom first AUG of p53 mRNA whereas �N-p53isoform is produced from an in-frame second AUGthat is 117 nucleotides downstream of first AUG(Figure 1). This isoform of p53 lacks transactivationdomain-I (TAD-I) which is essential for interactionof MDM2 E3 ubiquitin ligase.7 Elegantly designedassays were performed to rule out the possibilities ofalternate splicing, cryptic promoter activity, scanningand reinitiation within p53 mRNA using eithersynthetic bicistronic reporter constructs or p53cDNA containing constructs.7,46 Especially underdifferent stress conditions like chemical stresses thatinduce DNA damage, IR radiation, ER stress, FL-p53 and �N-p53 isoforms are translated by IRESmediated mechanism.47,48 Thapsigargin induced stresstriggers unfolded protein response and specificallyincreases �N-p53 isoform over the full lengthp53.47Interestingly in case of a human genetic disorderX-linked dyskeratosiscongenita (X-DC), aberrantIRES mediated p53 translation has been reported.49,50

It has also been reported that during oncogenic

5′ 3′5′UTR

1st IRES 2nd IRES1 134

p53

AUG AUG

ΔNp53

251

FIGURE 1 | Schematic representation of p53 mRNA showing twoIRES elements. The two AUG codons are depicted in p53mRNA, firstAUG positioned after 134nts responsible for full length p53 (FL-p53),second AUG positioned after 251nts responsible for �N-p53 synthesis.



induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs inp53 mRNA.50 Also, it has been demonstrated thatthe IRES responsible for �N-p53 translation is activeat G1/S phase while the IRES responsible for fulllength p53 translation is active at G2/M phase ofcell cycle.46

While the above discussion focussed on theimportance of 5′UTR in translation regulation ofp53, the importance of 3′UTR in the same cannotbe overlooked. 3′UTR contains two conserved U-rich sequences, located immediately upstream tothe polyadenylation site, resembling cytoplasmicpolyadenylation elements (CPE). These two CPEs inthe 3′UTR have been observed to regulate stabilityand translation of reporter mRNA in non-irradiatedas well as irradiated cells by binding to certain trans-acting factors. GAPDH, hnRNPD, and hnRNP A/Bbind specifically to p53 CPEs and could potentiallybe involved in the post translation regulation ofp53.51 Hzf and HuR interact independently with the3′UTR of p53 mRNA and facilitate the cytoplasmiclocalization of p53 mRNA in the presence of tumorsuppressor protein, ARF.52 RNPC1 inhibits p53translation by preventing cap binding protein eIF4Efrom binding to p53; however its binding to thepoly U element in the 3′UTR is required for thisinhibition.53 3′UTR mediated regulation of translationmight also depend on 5′–3′UTR interactions mediatedby different proteins. One of the well-studied modes ofinteraction is via PolyA binding protein (PABP) whichbridges eIF4G with the poly A tail of p53 mRNA.Recent reports suggest the existence of complementarysequences in the 5′ and 3′UTR of p53 mRNA enablingits circularization. The protein RPL26 facilitatesthis interaction thus regulating translation.54 Suchinteractions may also be mediated by trans-actingfactors binding to the IRES as seen in uncappedand polyadenylated RNA of encephalomyocarditisvirus.55 This highlights the importance of studyingITAFs binding to p53 mRNA which might shedlight on the proteins that help in 5′-3′UTRinteraction and thus translation regulation of p53isoforms.

Another mechanism of 3′UTR mediated p53translation regulation is through microRNAs. miR-125b, miR-504, miR-25, and miR-30d directly targetthe 3′UTR of p53 mRNA and down regulate p53protein levels, which results in reduced expressionof genes that are transcriptionally activated byp53.56 Therefore the mechanism underlying p53translation regulation involves interactions of proteinsand microRNAs with p53 5′UTR and 3′UTR as wellas 5′–3′UTR interactions (Figure 2(a) and (b)).

IRES

IRES

Ribosomes

5′UTR

Stress

3′UTR

Translation

Translation

Translation

Poly A binding protein(PABP)

miR-125b miR-504

Circularization5′-3′UTR interaction RPL 26 3′Poly A tail

5′m7 G cap

(a)

(b)

FIGURE 2 | (a) p53 translation regulation. ITAFs binding to theIRESs (purple: PTB, yellow: ANXA2, red: hnRNPC1/C2, and green: PSF)enhances translation. miRNAs binding to p53 3′UTR inhibits translation.(b) Circularization of p53 mRNA helps in translation. (1) Circularizationof p53 mRNA through PABP and cap of mRNA. (2) 5′–3′UTR interactionwith help of RPL26.

CIS ACTING ELEMENTS IN IRES RNATRANSLATION REGULATION

The function of IRES elements, depends oncoordinated interplay between important cis actingelements present within mRNA and cellular proteinsfunctioning in trans as critical trans-acting factors.42

Incidentally, very little conservation has been observedin the sequences and even secondary structures ofcellular IRES RNAs.41 Most of the cellular IRESelements are located within the 5′UTR region andupstream to the initiator AUG codon. However, thereare certain mRNAs in which the IRES are downstreamof the AUG codon, leading to production of truncatedprotein isoforms.57 To determine the structure of theIRES, enzymatic and chemical probing methods wereemployed. These complex structures include stemloops and pseudoknots.57 In the case of cellularIRESs, a partial deletion of IRES element does notchange in IRES activity drastically.26,58–61 It has beennoticed that in case of viral IRESs, small deletionsor point mutations in IRES element can lead todrastic change in IRES activity. However, silent pointmutations in coding sequence of p53 IRES like L22Land TSM alter the structure of RNA and decreaseIRES activity.62 A Single nucleotide polymorphism(SNP) C119T in 5′UTR region of p53 mRNA is inhuman melanoma cancer patients. This SNP leads todecrease in binding of one of the IRES-interactingtrans-acting factor (ITAF), PTB protein and there bydecrease in p53 IRES activity.63 These observations led



to the hypothesis that cellular IRESs contain multiplestructural modules and its activity is the combinationof these modules.

TRANS-ACTING FACTORS—p53 IRESMEDIATED TRANSLATIONREGULATION

Many reported cellular IRESs do not have a conservedsecondary structure but with the help of differentcellular trans-acting factors which act as RNAchaperones, can ultimately recruit the ribosome andinitiate the translation.61,64 These protein factors arealso known as IRES-interacting trans-acting factors(ITAFs). Many cellular ITAFs have been identifiedso far, these include PTB, hnRNPC2/ C2, HuR, Unr,PCBP1, La, DAP565 to name a few. PTB interacts withand controls the expression of Unr,66 p53,67 hIR68

(human insulin receptor), c-myc IRESs. hnRNPC1/C2 has been shown to interact with Unr,66 XIAP,66

p5367 IRESs. DAP5/p97 protein has been shown tointeract with BCl-2 and CDK1 IRESs and promotesthe IRES activity.69 In case of Bag-I IRES PCBP1 andPTB proteins bind to IRES RNA and unwind a specificregion by RNA chaperone activity. These changesfinally facilitate the recruitment of the ribosome.70,71

In the case of p53 IRES translation regulationof p53 mRNA is controlled by cis-acting elementsand trans-acting factors. Few cellular proteinsidentified for p53 mRNA translation such asPTB,67 hnRNPC1/C2,62 MDM2,9 RPL26,72 DAP5,73

Annexin A2, and PSF74 binds to p53 mRNA andpositively regulates translation; however RNPC153

and nucleolin72 negatively regulates p53 mRNAtranslation. In case of DNA damage RPL26 bindspreferentially to 5′UTR and enhances the translationand induces G1 cell cycle arrest. Recently itwas reported that hnRNPQ protein interacts withmouse p53 mRNA 5′UTR region and regulatesthe translation efficiency of p53 and consequently,levels of apoptosis.75 One of the study reported thatmouse p53 protein binds to its 5′UTR and negativelyregulates the translation of p53 mRNA, although themechanism of this translation initiation inhibition hasnot been explained.44

However, availability and compartmentalisationof these trans-acting factors plays a crucialrole in IRES mediated translation initiation65

(Figure 3). Under certain conditions, posttranslationalmodifications play a role in localisations of ITAFsand binding affinities for IRESs. In case of hnRNPA1,phosphorylation affects localisation as well as abilityto modulate the target IRESs activity, like c myc,cyclin D1, VEGF, XIAP, Apaf-1, and Unr mRNAs.76

In case of PTB it was reported that on doxorubicintreatment, it relocalizes from nucleus to cytoplasmand consequently there is an increase in p53 IRESactivity.65,67 Phosphorylation of the ITAF, hnRNPA1affects both its subcellular localization and abilityto modulate IRES activity of its targets. Undercellular stress conditions certain cellular mRNAsundergo IRES mediated translation to maintain theexpression of cell survival genes, for example theexpression of p53 and �N-p53 isoforms is regulatedby IRES mediated translation. This review highlightsthe factors that influence the IRES efficiency ofp53 mRNA. Differential expression of p53 and�N-p53 isoforms has impact on cell survival geneexpression and ultimately determines fate of thecell. It is yet to elucidate the mechanism of howtranslation initiates and is regulated from specificAUGs. Observations in two independent studiesreveal the growth suppressive effects of �Np53expression and its unique transcription targets. Crystal

DAP5

DAP5

RPL26

RPL26

IRES activity

p53 and ΔNp53expression

PTB

PTB

AnxA2

AnxA2

PSF

PSF

hnRNPC1/C2

hnRNPC1/C2

AUGAUG

AUGAUG

Genotoxic stress/cell cycle arrest

FIGURE 3 | Representation of proposed regulation of p53 IRESmediated translation. During stress and physiological conditions IREStrans-acting factors (green: DAP5, brown: hnRNPC1/C2, purple: PTB,yellow: ANXA2, and red: RPL26) come out from nucleus to cytoplasm toenhance IRES activity.



structure of IRES RNA would give more insightsinto understanding of the differential expressionof p53 isoforms that determines various functionslike stem cell differentiation, cell cycle arrest andapoptosis. Studying the mutations in p53 IRES andtheir effects on its activity will help in answeringdifferent important questions in many diseases andcancer. It has been shown by different groups, IRESinteracting proteins play a critical role in p53 IRESmediated expression. PTB, hnRNP C1/C2, MDM2,Annexin A2, and PSF proteins are well documented forregulations of IRES mediated translation. Availability

of these proteins in cytoplasm determines the p53 geneexpression. For instance PTB and MDM2 proteinsrelocalizes from nucleus to cytoplasm under genotoxicstress and enhance the p53 IRES activity. Not onlythe localization of ITAFs but also their expression andposttranslational modifications affects the p53 IRESactivity. It has been shown that ITAFs play a majorrole in IRES mediated translation of p53 isoforms. Itwould be interesting to identify different ITAFs whichregulate the differential expression of p53 isoformsand their mechanism of action.

ACKNOWLEDGMENT

We thank our laboratory members for their helpful discussion. This work is supported by Department ofBiotechnology (DBT) to S.D. S.A. is supported by pre-doctoral fellowship from Council of Scientific andIndustrial Research (CSIR), India.

REFERENCES1. Levine AJ, Oren M. The first 30 years of p53: growing

ever more complex. Nat Rev Cancer 2009, 9:749–758.

2. Junttila MR, Evan GI. p53—a Jack of all trades butmaster of none. Nat Rev Cancer 2009, 9:821–829.

3. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev1996, 10:1054–1072.

4. Vousden KH, Prives C. Blinded by the light: the growingcomplexity of p53. Cell 2009, 137:413–431.

5. Hermeking H. MicroRNAs in the p53 network:micromanagement of tumour suppression. Nat RevCancer 2012, 12:613–626.

6. Marcel V, Dichtel-Danjoy ML, Sagne C, Hafsi H, MaD, Ortiz-Cuaran S, Olivier M, Hall J, Mollereau B,Hainaut P, et al. Biological functions of p53 isoformsthrough evolution: lessons from animal and cellularmodels. Cell Death Differ 2011, 18:1815–1824.

7. Courtois S, Verhaegh G, North S, Luciani MG, LassusP, Hibner U, Oren M, Hainaut P. δN-p53, a naturalisoform of p53 lacking the first transactivation domain,counteracts growth suppression by wild-type p53.Oncogene 2002, 21:6722–6728.

8. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2promotes the rapid degradation of p53. Nature 1997,387:296–299.

9. Yin Y, Stephen CW, Luciani MG, Fahraeus R. p53Stability and activity is regulated by Mdm2-mediatedinduction of alternative p53 translation products. NatCell Biol 2002, 4:462–467.

10. Powell DJ, Hrstka R, Candeias M, Bourougaa K, Vojte-sek B, Fahraeus R. Stress-dependent changes in the

properties of p53 complexes by the alternative transla-tion product p53/47. Cell Cycle 2008, 7:950–959.

11. Candeias MM, Powell DJ, Roubalova E, Apcher S,Bourougaa K, Vojtesek B, Bruzzoni-Giovanelli H,Fahraeus R. Expression of p53 and p53/47 are con-trolled by alternative mechanisms of messenger RNAtranslation initiation. Oncogene 2006, 25:6936–6947.

12. Ungewitter E, Scrable H. δ40p53 controls the switchfrom pluripotency to differentiation by regulating IGFsignaling in ESCs. Genes Dev 2010, 24:2408–2419.

13. Maier B, Gluba W, Bernier B, Turner T, MohammadK, Guise T, Sutherland A, Thorner M, Scrable H.Modulation of mammalian life span by the shortisoform of p53. Genes Dev 2004, 18:306–319.

14. Ohki R, Kawase T, Ohta T, Ichikawa H, TayaY. Dissecting functional roles of p53 N-terminaltransactivation domains by microarray expressionanalysis. Cancer Sci 2007, 98:189–200.

15. Hinault C, Kawamori D, Liew CW, Maier B, Hu J,Keller SR, Mirmira RG, Scrable H, Kulkarni RN. δ40Isoform of p53 controls β-cell proliferation and glucosehomeostasis in mice. Diabetes 2011, 60:1210–1222.

16. Hofstetter G, Berger A, Berger R, Zoric A, Braicu EI,Reimer D, Fiegl H, Marth C, Zeimet AG, Ulmer H,et al. The N-terminally truncated p53 isoform δ40p53influences prognosis in mucinous ovarian cancer. Int JGynecol Cancer 2012, 22:372–379.

17. Takahashi R, Giannini C, Sarkaria JN, Schroeder M,Rogers J, Mastroeni D, Scrable H. p53 isoform profilingin glioblastoma and injured brain. Oncogene 2013,32:3165–3174.



18. Jang SK, Krausslich HG, Nicklin MJ, Duke GM,Palmenberg AC, Wimmer E. A segment of the 5′

nontranslated region of encephalomyocarditis virusRNA directs internal entry of ribosomes during in vitrotranslation. J Virol 1988, 62:2636–2643.

19. Pelletier J, Sonenberg N. Internal initiation oftranslation of eukaryotic mRNA directed by asequence derived from poliovirus RNA. Nature 1988,334:320–325.

20. Hellen CU, Sarnow P. Internal ribosome entry sitesin eukaryotic mRNA molecules. Genes Dev 2001,15:1593–1612.

21. Gradi A, Svitkin YV, Imataka H, Sonenberg N.Proteolysis of human eukaryotic translation initiationfactor eIF4GII, but not eIF4GI, coincides with theshutoff of host protein synthesis after poliovirusinfection. Proc Natl Acad Sci U S A 1998,95:11089–11094.

22. Lamphear BJ, Yan R, Yang F, Waters D, Liebig HD,Klump H, Kuechler E, Skern T, Rhoads RE. Mappingthe cleavage site in protein synthesis initiation factoreIF-4 γ of the 2A proteases from human Coxsackievirusand rhinovirus. J Biol Chem 1993, 268:19200–19203.

23. Lamphear BJ, Kirchweger R, Skern T, Rhoads RE.Mapping of functional domains in eukaryotic proteinsynthesis initiation factor 4G (eIF4G) with picornaviralproteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem 1995,270:21975–21983.

24. Gingras AC, Svitkin Y, Belsham GJ, Pause A, SonenbergN. Activation of the translational suppressor 4E-BP1following infection with encephalomyocarditis virusand poliovirus. Proc Natl Acad Sci U S A 1996,93:5578–5583.

25. Macejak DG, Sarnow P. Internal initiation oftranslation mediated by the 5′ leader of a cellularmRNA. Nature 1991, 353:90–94.

26. Stoneley M, Willis AE. Cellular internal ribosome entrysegments: structures, trans-acting factors and regulationof gene expression. Oncogene 2004, 23:3200–3207.

27. Bushell M, Poncet D, Marissen WE, Flotow H,Lloyd RE, Clemens MJ, Morley SJ. Cleavage ofpolypeptide chain initiation factor eIF4GI duringapoptosis in lymphoma cells: characterisation of aninternal fragment generated by caspase-3-mediatedcleavage. Cell Death Differ 2000, 7:628–636.

28. Mitchell SA, Spriggs KA, Bushell M, Evans JR, StoneleyM, Le Quesne JP, Spriggs RV, Willis AE. Identificationof a motif that mediates polypyrimidine tract-bindingprotein-dependent internal ribosome entry. Genes Dev2005, 19:1556–1571.

29. Fernandez J, Yaman I, Huang C, Liu H, LopezAB, Komar AA, Caprara MG, Merrick WC, SniderMD, Kaufman RJ, et al. Ribosome stalling regulatesIRES-mediated translation in eukaryotes, a parallel toprokaryotic attenuation. Mol Cell 2005, 17:405–416.

30. Gaccioli F, Huang CC, Wang C, Bevilacqua E,Franchi-Gazzola R, Gazzola GC, Bussolati O, SniderMD, Hatzoglou M. Amino acid starvation inducesthe SNAT2 neutral amino acid transporter by amechanism that involves eukaryotic initiation factor2α phosphorylation and cap-independent translation. JBiol Chem 2006, 281:17929–17940.

31. Yaman I, Fernandez J, Liu H, Caprara M, KomarAA, Koromilas AE, Zhou L, Snider MD, Scheuner D,Kaufman RJ, et al. The zipper model of translationalcontrol: a small upstream ORF is the switch thatcontrols structural remodeling of an mRNA leader.Cell 2003, 113:519–531.

32. Spriggs KA, Bushell M, Willis AE. Translationalregulation of gene expression during conditions of cellstress. Mol Cell 2010, 40:228–237.

33. Coldwell MJ, deSchoolmeester ML, Fraser GA,Pickering BM, Packham G, Willis AE. The p36 isoformof BAG-1 is translated by internal ribosome entryfollowing heat shock. Oncogene 2001, 20:4095–4100.

34. Gu L, Zhu N, Zhang H, Durden DL, Feng Y, ZhouM. Regulation of XIAP translation and inductionby MDM2 following irradiation. Cancer Cell 2009,15:363–375.

35. Sherrill KW, Byrd MP, Van Eden ME, Lloyd RE. BCL-2 translation is mediated via internal ribosome entryduring cell stress. J Biol Chem 2004, 279:29066–29074.

36. Fox JT, Stover PJ. Mechanism of the internalribosome entry site-mediated translation of serinehydroxymethyltransferase 1. J Biol Chem 2009,284:31085–31096.

37. Le SY, Maizel JV Jr. A common RNA structural motifinvolved in the internal initiation of translation ofcellular mRNAs. Nucleic Acids Res 1997, 25:362–369.

38. Pinkstaff JK, Chappell SA, Mauro VP, Edelman GM,Krushel LA. Internal initiation of translation of fivedendritically localized neuronal mRNAs. Proc NatlAcad Sci U S A 2001, 98:2770–2775.

39. Chappell SA, LeQuesne JP, Paulin FE, deSchoolmeesterML, Stoneley M, Soutar RL, Ralston SH, HelfrichMH, Willis AE. A mutation in the c-myc-IRES leadsto enhanced internal ribosome entry in multiplemyeloma: a novel mechanism of oncogene de-regulation. Oncogene 2000, 19:4437–4440.

40. Belin S, Beghin A, Solano-Gonzalez E, Bezin L,Brunet-Manquat S, Textoris J, Prats AC, MertaniHC, Dumontet C, Diaz JJ. Dysregulation of ribosomebiogenesis and translational capacity is associated withtumor progression of human breast cancer cells. PLoSOne 2009, 4:e7147.

41. Xia X, Holcik M. Strong eukaryotic IRESs have weaksecondary structure. PLoS One 2009, 4:e4136.

42. Martinez-Salas E, Ramos R, Lafuente E, Lopez deQuinto S. Functional interactions in internal translationinitiation directed by viral and cellular IRES elements. JGen Virol 2001, 82:973–984.



43. Kastan MB, Onyekwere O, Sidransky D, VogelsteinB, Craig RW. Participation of p53 protein in thecellular response to DNA damage. Cancer Res 1991,51:6304–6311.

44. Mosner J, Mummenbrauer T, Bauer C, Sczakiel G,Grosse F, Deppert W. Negative feedback regulationof wild-type p53 biosynthesis. EMBO J 1995,14:4442–4449.

45. Fu L, Benchimol S. Participation of the humanp53 3′UTR in translational repression and activationfollowing γ -irradiation. EMBO J 1997, 16:4117–4125.

46. Ray PS, Grover R, Das S. Two internal ribosome entrysites mediate the translation of p53 isoforms. EMBORep 2006, 7:404–410.

47. Bourougaa K, Naski N, Boularan C, Mlynarczyk C,Candeias MM, Marullo S, Fahraeus R. Endoplasmicreticulum stress induces G2 cell-cycle arrest via mRNAtranslation of the p53 isoform p53/47. Mol Cell 2010,38:78–88.

48. Halaby MJ, Yang DQ. p53 translational control: anew facet of p53 regulation and its implication fortumorigenesis and cancer therapeutics. Gene 2007,395:1–7.

49. Montanaro L, Calienni M, Bertoni S, Rocchi L,Sansone P, Storci G, Santini D, Ceccarelli C, TaffurelliM, Carnicelli D, et al. Novel dyskerin-mediatedmechanism of p53 inactivation through defectivemRNA translation. Cancer Res 2010, 70:4767–4777.

50. Bellodi C, Kopmar N, Ruggero D. Deregulation ofoncogene-induced senescence and p53 translationalcontrol in X-linked dyskeratosis congenita. EMBO J2010, 29:1865–1876.

51. Rosenstierne MW, Vinther J, Mittler G, Larsen L,Mann M, Norrild B. Conserved CPEs in the p533′ untranslated region influence mRNA stability andprotein synthesis. Anticancer Res 2008, 28:2553–2559.

52. Nakamura H, Kawagishi H, Watanabe A, Sugimoto K,Maruyama M, Sugimoto M. Cooperative role of theRNA-binding proteins Hzf and HuR in p53 activation.Mol Cell Biol 2011, 31:1997–2009.

53. Zhang J, Cho SJ, Shu L, Yan W, Guerrero T, Kent M,Skorupski K, Chen H, Chen X. Translational repressionof p53 by RNPC1, a p53 target overexpressed inlymphomas. Genes Dev 2011, 25:1528–1543.

54. Chen J, Kastan MB. 5′-3′-UTR interactions regulate p53mRNA translation and provide a target for modulatingp53 induction after DNA damage. Genes Dev 2010,24:2146–2156.

55. Mazumder B, Seshadri V, Fox PL. Translational controlby the 3′-UTR: the ends specify the means. TrendsBiochem Sci 2003, 28:91–98.

56. Kumar M, Lu Z, Takwi AA, Chen W, Callander NS,Ramos KS, Young KH, Li Y. Negative regulation of thetumor suppressor p53 gene by microRNAs. Oncogene2011, 30:843–853.

57. Komar AA, Lesnik T, Cullin C, Merrick WC, TrachselH, Altmann M. Internal initiation drives the synthesisof Ure2 protein lacking the prion domain and affects[URE3] propagation in yeast cells. EMBO J 2003,22:1199–1209.

58. Yang Q, Sarnow P. Location of the internal ribosomeentry site in the 5′ non-coding region of theimmunoglobulin heavy-chain binding protein (BiP)mRNA: evidence for specific RNA-protein interactions.Nucleic Acids Res 1997, 25:2800–2807.

59. Chappell SA, Edelman GM, Mauro VP. A 9-nt segmentof a cellular mRNA can function as an internal ribosomeentry site (IRES) and when present in linked multiplecopies greatly enhances IRES activity. Proc Natl AcadSci U S A 2000, 97:1536–1541.

60. Stoneley M, Paulin FE, Le Quesne JP, Chappell SA,Willis AE. C-Myc 5′ untranslated region containsan internal ribosome entry segment. Oncogene 1998,16:423–428.

61. Jopling CL, Spriggs KA, Mitchell SA, Stoneley M, WillisAE. L-Myc protein synthesis is initiated by internalribosome entry. RNA 2004, 10:287–298.

62. Grover R, Sharathchandra A, Ponnuswamy A, KhanD, Das S. Effect of mutations on the p53 IRES RNAstructure: implications for de-regulation of the synthesisof p53 isoforms. RNA Biol 2011, 8:132–142.

63. Khan D, Sharathchandra A, Ponnuswamy A, Grover R,Das S. Effect of a natural mutation in the 5′ untranslatedregion on the translational control of p53 mRNA.Oncogene 2013, 32:4148–4159.

64. Le Quesne JP, Stoneley M, Fraser GA, Willis AE.Derivation of a structural model for the c-myc IRES. JMol Biol 2001, 310:111–126.

65. Grover R, Candeias MM, Fahraeus R, Das S. p53and little brother p53/47: linking IRES activities withprotein functions. Oncogene 2009, 28:2766–2772.

66. Schepens B, Tinton SA, Bruynooghe Y, Parthoens E,Haegman M, Beyaert R, Cornelis S. A role for hnRNPC1/C2 and Unr in internal initiation of translationduring mitosis. EMBO J 2007, 26:158–169.

67. Grover R, Ray PS, Das S. Polypyrimidine tract bindingprotein regulates IRES-mediated translation of p53isoforms. Cell Cycle 2008, 7:2189–2198.

68. Spriggs KA, Cobbold LC, Ridley SH, Coldwell M,Bottley A, Bushell M, Willis AE, Siddle K. The humaninsulin receptor mRNA contains a functional internalribosome entry segment. Nucleic Acids Res 2009,37:5881–5893.

69. Marash L, Liberman N, Henis-Korenblit S, Sivan G,Reem E, Elroy-Stein O, Kimchi A. DAP5 promotes cap-independent translation of Bcl-2 and CDK1 to facilitatecell survival during mitosis. Mol Cell 2008, 30:447–459.

70. Pickering BM, Mitchell SA, Evans JR, Willis AE.Polypyrimidine tract binding protein and poly r(C)binding protein 1 interact with the BAG-1 IRES and



stimulate its activity in vitro and in vivo. Nucleic AcidsRes 2003, 31:639–646.

71. Pickering BM, Mitchell SA, Spriggs KA, Stoneley M,Willis AE. Bag-1 internal ribosome entry segmentactivity is promoted by structural changes mediatedby poly(rC) binding protein 1 and recruitment ofpolypyrimidine tract binding protein 1. Mol Cell Biol2004, 24:5595–5605.

72. Takagi M, Absalon MJ, McLure KG, Kastan MB.Regulation of p53 translation and induction after DNAdamage by ribosomal protein L26 and nucleolin. Cell2005, 123:49–63.

73. Weingarten-Gabbay S, Khan D, Liberman N, YoffeY, Bialik S, Das S, Oren M, Kimchi A. The

translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA. Oncogene (Epubahead of print; Jan 14, 2013).

74. Sharathchandra A, Lal R, Khan D, Das S. AnnexinA2 and PSF proteins interact with p53 IRES andregulate translation of p53 mRNA. RNA Biol 2012,9:1429–1439.

75. Kim DY, Kim W, Lee KH, Kim SH, Lee HR, KimHJ, Jung Y, Choi JH, Kim KT. hnRNP Q regulatestranslation of p53 in normal and stress conditions. CellDeath Differ 2013, 20:226–234.

76. Komar AA, Hatzoglou M. Cellular IRES-mediatedtranslation: the war of ITAFs in pathophysiologicalstates. Cell Cycle 2011, 10:229–240.


ires mediated translational regulation of p53 isoforms

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