Molecular Cancer Research - RNA-Binding Protein RBM24 … · 2014-03-07 · Chromatin, Gene, and...

Chromatin, Gene, and RNA Regulation

RNA-Binding Protein RBM24 Regulates p63 Expression viamRNA Stability

Enshun Xu, Jin Zhang, Min Zhang, Yuqian Jiang, Seong-Jun Cho, and Xinbin Chen

Abstractp63, a p53 family member, plays pivotal roles in epidermal development, aging, and tumorigenesis. Thus,

understanding how p63 expression is controlled has biological and clinical importance. RBM24 is an RNA-bindingprotein and shares a high sequence similarity with RBM38, a critical regulator of p63. In this study, we investigatedwhether RBM24 is capable of regulating p63 expression. Indeed, we found that ectopic expression of RBM24decreased, whereas knockdown of RBM24 increased, the levels of p63 transcript and protein. To explore theunderlying mechanism, we found that RBM24 was able to bind to multiple regions in the p63 30 untranslatedregion and, subsequently, destabilize p63 transcript. Furthermore, we showed that the 30 untranslated region in p63transcript and the RNA-binding domain in RBM24 were required for RBM24 to bind p63 transcript andconsequently, inhibit p63 expression. Taken together, our data provide evidence that RBM24 is a novel regulator ofp63 via mRNA stability.

Implications: Our study suggests that p63 is regulated by RBM24 via mRNA stability, which gives an insightinto understanding how posttranscriptional regulatory mechanisms contribute to p63 expression.Mol Cancer Res;12(3); 359–69. �2013 AACR.

Introductionp63 is a member of the p53 family, including p53, p63,

and p73 (1). All 3 proteins are transcriptional factors andshare a high sequence similarity, especially in the DNA-binding domain (2). Like other p53 family members, p63gene has complex expression patterns because of the usage of2 distinct promoters and alternative splicing at the C-terminus. The usage of 2 promoters results in 2 majorp63 isoforms, TAp63 and DNp63, and each isoform isalternatively spliced into at least 5 variants, a, b, g , d, ande (3, 4). Importantly, TAp63 isoforms, transcribed from theupstream P1 promoter, contain a transactivation domainsimilar to that in p53 and thus can induce a number of p53target genes including p21 and MDM2 (5). By contrast,DNp63 isoforms, transcribed from the P2 promoter inintron 3, lack the N-terminal transactivation domain andare presumably thought to be transcriptionally inactive.Interestingly, some studies showed that DNp63 carries aDN activation domain and retains transcriptional activityunder certain circumstances (2, 5, 6).

The biological function of p63 is complex because of thepresence of multiple isoforms with opposing functions.Studies suggest that the DNp63 isoforms have oncogenicpotential (7, 8), whereas the TAp63 isoforms play a role intumor suppression (9). This apparent conflict was recentlyaddressed by generating isoform-specific p63 knockoutmicemodels. Specifically, total p63 knockoutmice have defects inskin, teeth, mammary gland, and limb, and die soon afterbirth (10, 11), suggesting a critical role of p63 in epidermaldevelopment. Interestingly, mice deficient in DNp63 iso-forms largely phenocopy total p63 knockout mice (12).These mice die shortly after birth because of several devel-opmental defects, such as truncated forelimbs and theabsence of hind limbs. By contrast, mice deficient in TAp63isoforms are born live and tumor prone (9). In addition,thesemice develop several phenotypes, including acceleratedaging, obesity, insulin resistance, and glucose intolerance(13, 14). Together, these in vivo studies indicate a criticalrole of p63 in skin development, aging, metabolism, andtumorigenesis.Given the biological importance of p63, studies have been

carried out to elucidate howp63 expression is controlled. Forinstance, upon exposure to various stimuli, the level of p63transcript is regulated by p53 and several other transcriptionfactors (15–17). Moreover, p63 can be posttranscriptionallyregulated byRNA-binding protein (RBP) RBM38 andHuRvia mRNA stability and protein translation, respectively (18,19). In addition, several microRNAs (miRNA), includingmiR-302, miR-130b, and miR-203, are found to regulatep63 mRNA stability (20–22). Furthermore, p63 protein

Authors' Affiliation: Comparative Oncology Laboratory, University ofCalifornia at Davis, Davis, California

Corresponding Authors: Jin Zhang, Comparative Oncology Laboratory,University of California at Davis, Davis, CA 95616. Phone: 530-754-8408;Fax: 530-752-6042; E-mail: [email protected]; and Xinbin Chen,[email protected]

doi: 10.1158/1541-7786.MCR-13-0526

�2013 American Association for Cancer Research.

MolecularCancer

Research

www.aacrjournals.org 359

on October 17, 2020. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst December 27, 2013; DOI: 10.1158/1541-7786.MCR-13-0526

http://mcr.aacrjournals.org/

stability is regulated by a set of E3 ligases, such as itch, Pirh2,wwp1, and SCFbTrCP1 (23–26). Nevertheless, other regu-lators, which are critical in modulating p63 expression,remain to be elucidated.

Materials and MethodsReagentsAnti-RBM24, raised in rabbit, was generated by Cocalico

Biologicals. Anti-p63, 4A4, was purchased from Santa CruzBiotechnology. Anti-HA was purchased from Covance.Anti-actin, proteinase inhibitor cocktail, RNase A, andprotein A/G beads were purchased from Sigma. ScrambledsiRNA (GGC CGA UUG UCA AAU AAU U) and siRNAagainst RBM24 (CAC UGG AGC UGC AUA CGC A)were purchased from Dharmacon RNA Technologies.Transfection reagentMetafectene was purchased fromBion-tex (Germany). Silentfect lipid was purchased from Bio-RadLaboratories. TRizol reagent purchased from Invitrogen.The MMLV reverse transcriptase was purchased from Pro-mega. EST clone, containing a full-length human TAp63a(clone ID 5552611), was purchased from OpenBiosystem.

PlasmidsTo generate pcDNA3-HA-RBM24, a PCR product was

amplified by using cDNA samples from MCF7 cells as atemplate and then inserted into pcDNA3-HA vector viaEcoRI and XhoI sites. The primers were a forward primer, 50-GGGGAATTCATGCACACGACCCAGAAG-30, anda reverse primer, 50-GGG CTC GAG CTA TTG CATTCG GTC TGT CTG-30. To generate pcDNA3-RBM24,a PCR product was amplified by using cDNA samples fromMCF7 cells as a template and then inserted into pcDNA3vector viaHindIII andXhoI sites. The primers were a forwardprimer, 50-AAA AAG CTT CAC CAT GAT GCA CACGAC CCA GAA GGA CAC GAC GTA CA-30, and areverse primer, which is the same as the one for pcDNA3-HA-RBM24. To generate pcDNA4-RBM24 vector, aDNAfragment was digested from pcDNA3-HA-RBM24 andthen inserted into pcDNA4 vector via EcoRI and XhoI sites.To generate pcDNA3-RBM24-DRNP1 vector, a 2-step

PCR strategy was used. The first-step PCRwas performed toseparately amplify 2 DNA fragments by using RBM24 ESTclone as a template. Fragment 1was amplified with a forwardprimer, 50-AAA AAG CTT CAC CAT GAT GCA CACGAC CCA GAA GGA CAC GAC GTA CA-30, and areverse primer, 50-TCGGCAGCAGCCCGGTCAGCGGAC TTG CCC GTC TGC CGG TCG GTG-30. Frag-ment 2 was amplified with a forward primer, 50-ACC GGCAGACGGGCA AGTCCGCTG ACCGGGCTGCTGCCG AAA GGG-30, and a reverse primer, 50-GGG CTCGAG CTA TTG CAT TCG GTC TGT CTG-30. Thesecond-step PCRwas performed using amixture of fragment1 and 2 as a template with a forward primer, 50-AAA AAGCTT CAC CAT GAT GCA CAC GAC CCA GAA GGACACGACGTACA-30, and a reverse primer, 50-GGGCTCGAG CTA TTG CAT TCG GTC TGT CTG-30. ThisPCR product was then inserted into pcDNA3-RBM24

vector via HindIII and XhoI sites to generate pcDNA3-RBM24-DRNP1. To generate pcDNA3-RBM24-DRNP2vector, the same strategy was used with different primers.The primers to amplify fragment 1were a forward primer, 50-AAA AAG CTT CAC CAT GAT GCA CAC GAC CCAGAAGGACACGACGTACA-30, and a reverse primer, 50-GCGTCGGTGGTGTGGTAGGGCTTGGTGTACGTC GTG TCC T-30. The primers to amplify fragment 2were a forward primer, 50-AGGACACGACGTACACCAAGCCCTACCACACCACCGACGCCAGCC-30, anda reverse primer, 50-GGG CTC GAG CTA TTG CATTCG GTC TGT CTG-30. The primers for second-stepPCR were a forward primer, 50-AAA AAG CTT CAC CATGAT GCA CAC GAC CCA GAA GGA CAC GAC GTACA-30, and a reverse primer, 50-GGG CTC GAG CTATTG CAT TCG GTC TGT CTG-30.To generate pGEX-5X-1-RBM24 vector, a DNA frag-

ment was digested from pcDNA3-HA-RBM24 and theninserted into pGEX-5X-1 vector viaEcoRI andXhoI sites. Togenerate pGEX-5X-1-RBM24-DRNP1, a PCR product wasamplified by using pcDNA3-RBM24-DRNP1 as a templateand then inserted into pGEX-5X-1 via EcoRI and XhoI sites.The PCR primers were a forward primer, 50-CGG AATTCA TGC ACA CGA CCC AGA AGG ACA CGA CGTACA-30 and a reverse primer, 50-GGG CTC GAG CTATTG CAT TCG GTC TGT CTG-30. To generate pGEX-5X-1-RBM24-DRNP2, the same strategy was used exceptthat pcDNA3-RBM24-DRNP2 was used as a template.To generate pcDNA3-TAp63a expression vector, a DNA

fragment were amplified by using TAp63a EST clone as atemplate with a forward primer, 50-GGG GAA GCT TGCCAC CAT GAA TTT TGA AAC TTC ACG G-30, and areverse primer, 50-GGG GGA TCC TCA CTC CCC CTCCTC TTT GAT G-30. The PCR products were cloned intopcDNA3 via HindIII and BamHI sites. To generatepcDNA3-TAp63a-30UTR expression vector, a DNA frag-ment containing the full-length p63 30UTR was amplifiedby using TAp63a EST clone as a template and then insertedinto pcDNA3-TAp63a vector via BamHI and XhoI sites.The PCR primers were a forward primer 50-GGG GGATCC GCC TCA CCA TGT GAG CTC TTC C-30 and areverse primer, 50-GGG GCT CGA GCA ATT TCT TAATTA GTT TTT ATT TAT TTT TTA AAT TTT ATTGCA TGT CCT GGC AAA CAA AAA GAG-30.

Cell culture and cell line generationHaCaT, ME180, MIA-PaCa2, MCF10A, HCT116, and

MCF7 cells were cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with 10% FBS as previ-ously described (18, 27). Mouse Embryo Fibroblasts(MEFs) were cultured in DMEM supplemented with10% FBS, 1� nonessential amino acids, and 55 mmol/Lb-mercaptoethanol. HCT116 and MCF7 stable cell linesthat can inducibly express RBM24 were generated asdescribed previously (28). Briefly, pcDNA4-RBM24 wastransfected into HCT116 and MCF7 parental cells, whichexpress a tetracycline repressor (pcDNA6; ref. 28). TheRBM24 expressing cells were selected with zeocin (150

Xu et al.

Mol Cancer Res; 12(3) March 2014 Molecular Cancer Research360




mg/mL) and confirmed by Western blot analysis. To induceRBM24 expression, tetracycline (1 mg/mL) was added tomedium for various times.

MEF isolationRBM38�/�; p53�/� MEFs were isolated as previously

described (29). To generate TAp63�/� MEFs, mice hetero-zygous for TAp63 (14), a gift from Dr. Elsa R. Flores' lab,were bred. MEFs were isolated from 13.5-day-old embryosas previously described (30). All animals are housed at theUniversity of California at Davis CLAS vivarium facility. Allanimals and use protocols were approved by the Universityof California at Davis Institution Animal Care and UseCommittee.

Western blot analysisWestern blot analysis was performed as previously

described (5). Briefly, cells lysates were collected and resus-pended with 1� SDS sample buffer. Proteins were then

resolved in an 8% to 12% SDS-PAGE gel and transferred toa nitrocellulose membrane, followed by ECL detection. Thelevel of protein was quantified by densitometry. The data arerepresentative of 3 independent experiments.

Recombinant protein purification, RNA probegeneration, and REMSABacteria BL21 was transformed with a pGEX-5X-1 vector

expressing glutathione S-transferase (GST)-tagged RBM24,DRNP1, or DRNP2 and positive clones were selected. Therecombinant proteins were then purified by glutathionesepharose beads (Amersham Biosciences). RNA probes con-taining various regions of p63 30UTR were generated aspreviously described (18). The p21 probe was generated aspreviously described (31). RNA electrophoretic mobilityassay (REMSA) was performed as described previously(18). Briefly, 32P-labeled probes were incubated with recom-binant protein in a binding buffer [10 mmol/L HEPES-KOH at pH 7.5, 90mmol/L potassium acetate, 1.5mmol/L

A

B C D EME180

Sequence alignment between human Rbm38 and Rbm24 proteins

Identities 160/238 (67%); positive 170/238 (72%); Gaps 24/238 (10%)

1.0 0.5

Actin Actin Actin

1.0 0.5 1.0 0.5 1.0 0.5

RB

M24

ctr

l

RB

M24

ctr

l

RB

M24

ctr

l

RB

M24

ctr

l

HaCaT MCF10A MIA-PaCa-2

RBM24

(α-HA)

ΔNp63α ΔNp63α TAp63α

RBM24

(α-HA)

RBM24

(α-HA)

Figure 1. Ectopic expression of RBM24 suppresses p63 expression. A, sequence similarity between human RBM38 and RBM24. The RNP1 and RNP2submotifs are shown in boxes. B toD, ectopic expression of RBM24 inhibitsDNp63a expression.ME180 (B), HaCaT (C), andMCF10A (D) cellswere transientlytransfected with a control vector or a vector expressing HA-tagged RBM24 for 48 hours, and the level of HA-tagged RBM24, DNp63a, and actin wasdetermined by Western blot analysis. The level of DNp63a protein was normalized to that of actin and arbitrary set as 1.0 in control cells. The relative foldchanges were shown below each lane. E, RBM24 inhibits TAp63a expression. MIA-PaCa2 cells was transiently transfected with a control vector or a vectorexpressing HA-tagged RBM24 for 48 hours, followed by Western blot analysis to determine the level of HA-tagged RBM24, TAp63a, and actin. The level ofTAp63a protein was normalized to that of actin and arbitrary set as 1.0 in control cells. The relative fold changes were shown below each lane.

p63 Expression Is Regulated by RBM24 through mRNA Stability

www.aacrjournals.org Mol Cancer Res; 12(3) March 2014 361




magnesium acetate, 2.5 mmol/L dithiothreitol, 40 U ofRNase inhibitor (Ambion)] at 30�C for 30 minutes. RNA–protein complexes were resolved on a 6% acrylamide gel andradioactive signals were detected by autoradiography.

RNA isolation and reverse transcription-PCR analysisTotal RNAswere isolated using TRizol reagent. cDNAwas

synthesized using MMLV reverse transcriptase according tothe user's manual. The PCR program used for amplificationwas (i) 94�Cfor 5minutes, (ii) 94�Cfor 45 seconds, (iii) 58�Cfor 45 seconds, (iv) 72�C for 1 minute, and (v) 72�C for 10minutes. From steps 2 to 4, the cyclewas repeated 20 times forhuman andmouse actin or 30 times for RBM24 and P63. Toamplify human actin, 2 pairs of primers were used. The firstpair of primers was used for the RT-PCR analysis in Figs. 2Aand C, 3A, and 4A whereas the second pair of primers wereused for all other RT-PCR analysis. The first pair of primerswere forward primer, 50-CTG AAG TAC CCC ATC GAGCAC GGC A-30, and reverse primer, 50-GGA TAG CACAGC CTG GAT AGC AAC G-30. The second pair ofprimers were forward primer, 50-AGC GCG GCT ACAGCTTCA-30, and reverse primer, 50-CGTAGCACAGCTTCTCCTTAA TGTC-30. These primers were used for therest of RT-PCR analysis. The primers to amplify mouse actinwere forward primer, 50-CCC ATC TAC GAG GGC TAT-30, and reverse primer, 50-AGAAGGAAGGCTGGAAAA-30. The primers to amplify human RBM24 were forwardprimer, 50-AGC CTG CGC AAG TAC TTC G-30, andreverse primer, 50-CAG GCC CTT TCG GCA G-30. Theprimers to amplify mouse RBM24 were forward primer, 50-ACCCAGAAGGACACGACGTA-30, and reverse primer,50-TCG ATG ATG GGG TTG GGA T-30. The primers toamplify human DNp63 were forward primer, 50-TACCTG GAA AAC AAT GCC-30, and reverse primer, 50-ACT GCT GGA AGG ACA CG-30. The primers toamplify human TAp63 were forward primer, 50-AGCCCA TTG ACT TGA ACT T-30, and reverse primer,50-GGA CTG GTG GAC GAG GA-30. The primers toamplify mouse DNp63 were forward primer, 50-TACCTG GAA AAC AAT GCC CA-30, and reverse primer,50-GCT GGA AGG ACA CAT CGA A-30. To measurethe precursor mRNA, total RNAs were isolated usingTRizol reagent and then treated with DNase I to removegenomic DNA before cDNA synthesis. The primers forhuman p63 pre-mRNA were forward primer, 50-CTTGTT GTT AAC AAC AGC ATG AG-30, and reverseprimer, 50-AGA AAG CCT GTG CCA CTC AC-30.Quantitative PCR (qPCR) was performed using 2�

qPCR SYBR Green Mix (ABgene) with 5 mmol/L primers.Reactions were run on a realplex using a 2-step cyclingprogram: 95 �C for 15 minutes, followed by 40 cycles of95�C for 15 seconds, 60�C for 30 seconds, 68�C for 30seconds. A melting curve (57–95�C) was generated at theend of each run to verify the specificity. The primers forhuman actin were forward primer, 50-TCCATCATGAAGTGT GAC GT-30, and reverse primer, 50-TGA TCC ACATCT GCT GGA AG-30. The primers for human TAp63were forward primer, 50-AGC CCA TTG ACT TGA ACT

T-30, and reverse primer, 50-GGA CTG GTG GAC GAGGA-30. The primers to amplify human DNp63 were forwardprimer, 50-TACCTGGAAAAC AATGCC-30, and reverseprimer, 50-ACT GCT GGA AGG ACA CG-30.

RNA-Chip analysisRNA-Chip analysis was performed as previously described

(32). Briefly, cells (2 � 107) were lysed with an immuno-precipitation buffer (100 mmol/L KCl, 5 mmol/L MgCl2,10mmol/LHepes, 1 mmol/L DTT, and 0.5%NP-40), andthen incubated with 2 mg of anti-RBM24 or rabbit immu-noglobulin G (IgG) at 4�C overnight. The RNA–proteinimmunocomplexes were brought down by protein A/Gbeads, followed by RT-PCR analysis.

A B

C D

E F

ME180 ME180

HaCaT HaCaT

MIA-PaCa-2 MIA-PaCa-2

Actin Actin

ActinActin

RBM24

RBM24 siRNAScr

RBM24 siRNAScr

RBM24 siRNAScr RBM24 siRNAScr

RBM24 siRNAScr

RBM24 siRNAScr

RBM24

Actin Actin

RBM24

1.0 0.5

1.0 0.4 1.0 1.5

1.0 2.0

1.0 1.61.0 0.3

ΔNp63α

ΔNp63α

TAp63α

Figure 2. Knockdown of RBM24 increases p63 expression. A, C, ME180(A) and HaCaT (C) cells were transiently transfected with a control orRBM24 siRNA for 72 hours, followed by RT-PCR analysis to determinethe level of RBM24 and actin transcripts. The level of RBM24 transcriptwas normalized to that of actin and arbitrary set as 1.0 in control cells. Therelative fold changes were shown below each lane. Band D, ME180 (B)and HaCaT (D) cells were treated as described in A, and the level ofDNp63a and actin proteinswas determined byWestern blot analysis. Thelevel of DNp63a protein was normalized to that of actin protein andarbitrary set as 1.0 in control cells. The relative fold changes were shownbelow each lane. E, MIA-PaCa2 cells were transiently transfected with acontrol or RBM24 siRNA for 72 hours and the level of RBM24 and actintranscripts was determined by RT-PCR analysis. F, RBM24 knockdownincreases TAp63a expression.MIA-PaCa2 cells were treated as describein (E), and the level of TAp63a and actin proteins was determined byWestern blot analysis. The level of TAp63a protein and transcript werenormalized to that of actin and arbitrary set as 1.0 in control cells. Therelative fold changes were shown below each lane.

Xu et al.





Statistical analysisAll experiments were performed at least 3 times. Numer-

ical data were expressed as mean � SDs. Two groupcomparisons were analyzed by 2-sided Student t test. Pvalues were calculated, and a P of <0.05 was consideredsignificant.

ResultsEctopic expression of RBM24 suppresses, whereasknockdown of RBM24 increases, p63 expressionIn an effort to understand the underlying mechanisms by

which p63 expression is controlled, we showed previouslythat RBM38, also called RNPC1, is able to destabilize p63

transcript and plays a critical role in p63-mediated kerati-nocyte differentiation (18). Interestingly, a search of genedatabase revealed that RBM38 has a paralogue, namedRBM24, which shares a high degree of sequence similaritywith that of RBM38 (Fig. 1A). The RBM24 gene encodes236 aa and is located on chromosome 6. Structure analysisshows that RBM24 contains one RNA-binding domain,which is composed of 2 submotifs, RNP1 and RNP2. Mostremarkably, the RNA-binding domain in RBM24 is iden-tical to the one inRBM38 (Fig. 1A). Therefore, it is plausiblethat RBM24 may regulate p63 expression.To determine whether RBM24 regulates p63 expression,

a control vector or a vector expressing HA-tagged RBM24was transiently transfected into ME180 cells. The level of

ME180 HaCaT

MIA-PaCa-2

MCF10A

RB

M2

4

Re

lative

fold

ch

an

ge

of

ΔN

p6

3 t

ran

scri

pt

Re

lative

fold

ch

an

ge

of

ΔN

p6

3 t

ran

scri

pt

Re

lative

fold

ch

an

ge

of

TA

p6

3 t

ran

scri

pt

ctr

l

RB

M2

4

ctr

l

RBM24Ctrl

RBM24Ctrl

1

0.6

0.2

0

1

0.6

0.2

0

1

0.6

0.2

RBM24Ctrl

RB

M2

4

ctr

l

RB

M2

4

ctr

l

RB

M2

4

ctr

l

1.0 0.5

1.0 0.5

1.0 0.5 1.0 0.1

1.0 0.1

1.0 0.6 1.0 0.3

Actin Actin Actin

RBM24 RBM24

MCF7-RBM24

RNPC1–/–;P53–/–

HCT116-RBM24

RBM24

RBM24– + – +

A B C

F G

I J

H

D E

ΔNp63

Actin

RBM24

Actin

RBM24

ΔNp63

ΔNp63 ΔNp63

TAp63

Figure 3. The level of p63 transcript is decreased by ectopic expression of RBM24. A–C, F, ME180 cells (A), HaCaT cells (B), MCF10A cells (C), andRBM38�/�;p53�/� MEFs (F) were transiently transfected with a control vector or a vector expressing HA-tagged RBM24 for 48 hours. Total RNAs were isolated andsubjected to RT-PCR analysis to determine the level of RBM24, DNp63, and actin transcripts. The level of DNp63 transcript was normalized to that ofactin and arbitrary set as 1.0 in control cells. The relative fold changeswere shown below each lane. D and E, HCT116 (D) andMCF7 (E) cells were uninduced orinduced to express RBM24 for 48 hours. Total RNAs were isolated and subjected to RT-PCR analysis to determine the level of RBM24, DNp63, andactin transcripts. G, the level of DNp63 transcript in HaCaT cells, which were transfected with a control vector or RBM24-expressing vector, was measured byqRT-PCR.The level of actinmRNAwasmeasured asan internal control. H, the experimentwasperformedas inGexcept thatMCF7cells,whichwere uninducedor induced to express RBM24, were used. I–J,MIA-PaCa2 cells were transiently transfectedwith a control vector or a vector expressing HA-taggedRBM24 for48 hours. Total RNAs were isolated and subjected to RT-PCR analysis (I) or qRT-PCR (J) to determine the level of RBM24, TAp63, and actin transcripts.






RBM24 was detectable upon transfection (Fig. 1B, RBM24panel). Interestingly, we found that the DNp63a proteinwas markedly inhibited by RBM24 (Fig. 1B, DNp63apanel). Similarly, we found that RBM24 inhibited DNp63aexpression in HaCaT and MCF10A cells (Fig. 1C and D,DNp63a panels). Furthermore, we tested whether RBM24has an effect on TAp63 expression by using MIA-PaCa2cells, in which TAp63a is highly expressed (27). We foundthat the level of TAp63a protein was markedly decreased byectopic expression of RBM24 (Fig. 1E, TAp63a panel).Together, these data suggest that p63 expression is repressedby ectopic expression of RBM24.To determine whether endogenous RBM24 regulates p63

expression, ME180 and HaCaT cells were transiently trans-fected with a control siRNA or a siRNA against RBM24.Again, we found that the level of RBM24 transcript wasmarkedly reduced by RBM24, but not by control, siRNA(Fig. 2A andC, RBM24 panels). Importantly, we found thatthe level of DNp63a proteins was increased by RBM24knockdown (Fig. 2B andD,DNp63a panels). Furthermore,we tested whether TAp63a expression is regulated byendogenous RBM24 and found to be increased uponRBM24 knockdown in MIA-PaCa2 cells (Fig. 2E and F).Together, these data suggest that knockdown of RBM24increases p63 expression.

Ectopic expression of RBM24 decreases, whereasknockdown of RBM24 increases, the level of p63transcriptRBPs are known to posttranscriptionally regulate their

targets mainly through mRNA stability or protein transla-tion. Thus, to explore how RBM24 regulates p63 expres-sion, the level of p63 transcript wasmeasured inME180 cellstransiently transfected with a control or RBM24 expressionvector.We found that upon transient expression of RBM24,the level of DNp63 transcript was decreased in ME180 cells(Fig. 3A, DNp63 panel). Similarly, ectopic expression ofRBM24 was able to reduce the level of DNp63 transcript inHaCaT andMCF10A cells (Fig. 3B and C, DNp63 panels).To verify this, HCT116 and MCF7 cells that can induciblyexpress RBM24 were used. We found that the level ofDNp63 transcript was decreased upon RBM24 induction(Fig. 3D and E, DNp63 panels). Next, we determinedwhether RBM24 regulates p63 expression in the absenceof p53 and RBM38. To address this, RBM38�/�;p53�/�

MEFs were transiently transfected with a control or RBM24expression vector and the level of p63 transcript was mea-sured. We found that RBM24 was able to significantlydecrease the level of p63 transcript in the absence of p53and RBM38 (Fig. 3F, DNp63 panel). Consistently, qPCRanalysis showed that the level of DNp63 transcript wasdecreased by ectopic expression of RBM24 in HaCaT andMCF7 cells (Fig. 3G and H). Furthermore, we determinedwhether RBM24 regulates TAp63 transcript by RT-PCRand qPCR. We showed that the level of TAp63 transcriptwas markedly decreased by ectopic expression of RBM24 inMIA-PaCa2 cells (Fig. 3I–J). Together, these data suggest

that ectopic expression of RBM24 decreases the level of p63transcript.Next, to determine whether endogenous RBM24 regu-

lates p63 transcript, ME180 and HaCaT cells were tran-siently transfected with a control siRNA or a siRNA againstRBM24. We found that the level of DNp63 transcript wasincreased by RBM24 knockdown (Fig. 4A and B, DNp63panels). Likewise, knockdown of RBM24 resulted inincreased levels of DNp63 transcript in RBM38�/�;p53�/�

MEFs (Fig. 4C, DNp63 panel). Furthermore, we found thatthe level of TAp63 transcript in MIA-PaCa2 cells was increas-ed by RBM24 knockdown (Fig. 4D and E). Together, thesedata suggest that the level of p63 transcript is increased byRBM24 knockdown.

RBM24 destabilizes p63 transcriptTo investigate the underlying mechanism by which

RBM24 regulates p63 expression, we first determinedwhether RBM24 regulates p63 transcription. Specifically,the level of p63 pre-mRNA was measured in HaCaT cells,transiently transfected with a control or RBM24 expressionvector. We found that overexpression of RBM24 had noeffect on the level of p63 pre-mRNA in HaCaT cells (Fig.5A, pre-p63 panel). Consistent with this, the level of p63pre-mRNAwas not altered by ectopic expression of RBM24

A B

D E

CME180 HaCaT RNPC1–/–;p53–/–

Actin

RBM24

Actin

RBM24

1.6

1

0

ΔNp63

Actin

RBM24

ΔNp63

Actin

RBM24

ΔNp63

MIA-PaCa-2

TAp63

RBM24 siRNAScr RBM24 siRNAScr RBM24 siRNAScr

RBM24 siRNAScr

RBM24-KDCtrl

Re

lative

fo

ld c

ha

ng

e o

f

TA

p6

3 t

ran

scri

pt

1.0 0.5 1.0 0.5

1.0 0.5

1.0 1.5

1.0 1.6

1.0 0.4

1.0 1.71.0 2.0

Figure4. KnockdownofRBM24 increases the level of p63 transcript. A–C,ME180 cells (A), HaCaT cells (B), and RBM38�/�;p53�/� MEFs (C) weretransiently transfected with a control or RBM24 siRNA for 72 hours. TotalRNAs were purified and subjected to RT-PCR analysis to determine thelevel of RBM24, DNp63, and actin transcripts. The level of DNp63transcriptwas normalized to that of actin and arbitrary set as 1.0 in controlcells. The relative fold changeswere shown below each lane. D and E, thelevel of TAp63 transcript is increased by knockdown of RBM24. MIA-PaCa2 cells were transiently transfected with a control or RBM24 siRNAfor 72 hours. Total RNAs were purified and subjected to RT-PCR (D) orqRT-PCR (E) to determine the level of RBM24, TAp63, and actintranscripts. The level of TAp63 transcript was normalized to that of actinand arbitrary set as 1.0 in control cells. The relative fold changes wereshown below each lane.

Xu et al.





in ME180 and MIA-PaCa2 cells (Fig. 5B and C, pre-p63panels). Similarly, RBM24 knockdown had no effect on p63pre-mRNA in HaCaT, ME180, and MIA-PaCa2 cells (Fig.5D–F, pre-p63 panels). These results suggest that p63 is

regulated by RBM24 via posttranscriptional mechanisms,such as mRNA stability. Thus, the half-life of DN and TAp63 transcripts was measured in cells treated with actino-mycinD,which inhibits de novoRNA synthesis. Specifically,

ME180HaCaT ME180HaCaTMIA-PaCa-2 MIA-PaCa-2

MIA-PaCa-2

RB

M24

Rela

tive

perc

enta

ge o

f

ΔNp63 tra

nscri

pts

Rela

tive

perc

enta

ge o

f

TA

p63 tra

nscri

pts

ctr

l

RB

M24

RB

M24

ctr

l

Scr

RB

M24

Scr

RB

M24

Scr

RBM24

Ctrl T1/2=3.7 h

T1/2=2.7 h RBM24

Ctrl T1/2=9.3 h

T1/2=7.2 h

1.2

1.0

0.8

0.6

0.4

0.2

1.2

1.0

0.8

0.6

0.4

0.2

RBM24Ctrl

0 1 2 3 4 5

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 89 10 11 12

0 1 2 3 4 5 Act D (h) 0 4 8 12 0 4 8 12 Act D (h)

RBM24Ctrl

RB

M24

ctr

l

1.0 1.1 1.0 1.0 1.0 1.0 1.0

1.0 0.4 1.0 0.5 1.0 0.5

1.0 1.0 1.01.11.1

0 0 4 8 12 h1 2 3 4 5 6 h

Actin

Actin Actin

RBM24

MCF7-RBM24

A

G H

B C D E F

pre-p63

Actin

RBM24

pre-p63

Actin

RBM24

siRNA siRNA

pre-p63

Actin

RBM24

pre-p63

TAp63ΔNp63

Figure 5. RBM24 destabilizes p63 transcript. A–C, ectopic expression of RBM24 has no effect on the level of p63 pre-mRNA. HaCaT (A), ME180 (B),andMIA-PaCa2 (C) cellswere transiently transfectedwith a control vector or a vector expressingRBM24 for 48 hours. Total RNAswere isolated and subjectedto RT-PCR analysis to determine the level of p63 pre-mRNA, RBM24, and actin mRNA. The level of p63 pre-mRNA was normalized to that of actin andarbitrary set as 1.0 in control cells. The relative fold changes were shown below each lane. D–F, knockdown of RBM24 has no effect on the level ofp63 pre-mRNA. HaCaT (D), ME180 (E), and MIA-PaCa2 (F) cells were transiently transfected with a control or RBM24 siRNA for 72 hours. Total RNAs werepurified and subjected to RT-PCR analysis to determine the level of RBM24 and actin mRNA as well as p63 pre-mRNA. The level of p63 pre-mRNA orRBM24 mRNA was normalized to that of actin and arbitrary set as 1.0 in control cells. The relative fold changes were shown below each lane.G, RBM24 shortens the half-life of DNp63 transcript. Top, MCF7 cells were uninduced or induced to express RBM24 for 48 hours, followed by treatmentwith 7 mg/mL actinomycin D for various times. Total RNAs were isolated and then subjected to RT-PCR analysis to determine the level of DNp63and actin transcripts. Bottom, the level ofDNp63 transcript was normalized to that of actin and plotted alongwith a time course to calculate the relative half-lifeof DNp63 mRNA. H, RBM24 shortens the half-life of TAp63 transcript. Top, MIA-PaCa2 cells were transiently transfected with a control vector orRBM24-expressing vector for 48 hours, followed by treatment with 7 mg/mL actinomycin D for various times. Total RNAs were isolated and then subjected toRT-PCR analysis to determine the level of TAp63 and actin transcripts. Bottom, the level of TAp63 transcript was normalized to that of actin and plotted alongwith a time course to calculate the relative half-life of TAp63 mRNA.






MCF7 cells were uninduced or induced to express RBM24for 48 hours, followed by actinomycin D treatment forvarious times. Similarly, MIA-PaCA2 cells were transfectedwith a control vector or a vector expressing RBM24, fol-lowed by actinomycin D treatment for various times. Thelevel of DN and TA p63 transcripts was determined by RT-PCR analysis and the relative half-life of DN and TA p63transcripts was calculated. We showed that the half-life ofDNp63 mRNA was deceased from 3.7 hours in the controlcells to 2.7 hours in cells with RBM24 expression (Fig. 5G),and the half-life of TAp63 mRNA was deceased from 9.3hours in the control cells to 7.2 hours in cells with RBM24expression (Fig. 5H). Together, these data suggest thatRBM24 shortens the half-life of p63 mRNA.

RBM24 binds to multiple regions in the 30UTR of p63transcriptTo further decipher the underlying mechanism by which

RBM24 destabilizes p63 mRNA, we determined whether

RBM24 associates with p63 transcript in vivo by performingan RNA-Chip analysis. We found that p63 transcript waspresent in RBM24, but not control IgG, immunoprecipi-tates (Fig. 6A, compare lane 2 with lane 3). As a control,RBM24 was unable bind to actin mRNA (Fig. 6A, lane 3).Next, the binding site(s) of RBM24 in p63 transcript wasmapped by performing REMSA. Specifically, radiolabeledprobes (A–C), spanning the entire p63 30UTR (Fig. 6B),were incubated with recombinant GST or GST-taggedRBM24 protein, followed by electrophoresis. We foundthat the recombinant GST-tagged RBM24, but not GSTprotein, formed a complex with probes A and C, but notprobe B (Fig. 6C, compare lanes 1, 4, and 7 with 2, 5, and 8,respectively). Importantly, these RNA-RBM24 complexeswere further disrupted by cold probe derived from p2130UTR (Fig. 6C, lanes 3, 6, and 9), which is known tocarry an AU-rich element.To further verify that the p63 30UTR is required for

RBM24 to inhibit p63 expression, we generated a TAp63a

HCT116-RBM24

A B

C D E

RBM24ctrl RBM24ctrl

RBM24

1 2 3

1 90 2132

CDS

A

AU-rich

3′UTR-A 3′UTR-B

3′UTR

3′UTR-C

U-rich

B C

3000 4010 4932

1 2 3 4 5 6 7 8 9

1.0 0.9 1.0 0.3

RBM24

GST

RPC

Cold p21

IP:R

BM

24

IP:IgG

Input

Actin

Actin

p63

TAp63α

TAp63α−CDS TAp63α−CDS

RBM24

Actin

TAp63α

Figure 6. RBM24 binds to multiple regions in the 30UTR of p63 transcript. A, RBM24 associates with p63 transcript in vivo. RBM24-expressing HCT116 cellextracts were immunoprecipitated with a control IgG or RBM24 antibody to bring down the protein–RNA complex. Total RNAs were isolated and subjectedto RT-PCR analysis to measure the level of p63 and actin transcripts. Five percent of cell lysate was used as input. B, schematic representation of p63transcript and the location of probes used for REMSA. The AU- or U-rich elements were shown in shaded boxes. C, RBM24 binds to multiple regions inp63 30UTR. REMSA was performed by mixing recombinant GST or GST-fused RBM24 protein with 32P-labeled probe A, B, or C. The bracket indicatesRNA–protein complexes. For competition assay, unlabeled p21 probe was added to the reaction mix before incubation with the 32P-labeled probeA, B, or C. D–E, the p63 30UTR is required for RBM24 to inhibit TAp63a expression. TAp63�/�MEFs were cotransfected with a control or RBM24-expressingvector along with a TAp63a expression vector that contains the coding region alone (D) or in combination with a full-length p63 30UTR (E). Cell lysates werecollected and the level of RBM24, TAp63a, and actin proteinswas determined byWestern blot analysis. The relative level of TAp63awas normalized to that ofactin and arbitrary set as 1.0 in control cells. The relative fold changes were shown below each lane.

Xu et al.





expression vector that contains TAp63a coding sequencealone or together with a full-length p63 30UTR. Next, theseTAp63a expression vectors were transiently transfected intoTAp63�/�MEFs along with a control or RBM24 expressionvector. We found that RBM24 had no effect on TAp63aexpression from an expression vector that only containsTAp63a coding sequence (Fig. 6D). By contrast, TAp63aexpression was significantly inhibited by RBM24 when theTAp63a expression vector contains a full-length p63 30UTR(Fig. 6E). Together, these results suggest that the p6330UTR is necessary for RBM24 to repress p63 expression.

The RNA-binding domain is required for RBM24 toinhibit p63 expressionThe RNA-binding domain in RBM24 is composed of 2

RNA recognition submotifs, RNP1 and RNP2 (Fig. 7A).Thus, to determine whether the RNA-binding domain isrequired for RBM24 to inhibit p63 expression, we generated2RBM24deletionmutants, which lackRNP1(DRNP1) andRNP2 (DRNP2), respectively (Fig. 7A). Next, the ability ofDRNP1 or DRNP2 to bind to p63 30UTR was determinedby REMSA. We found that neither DRNP1 nor DRNP2were capable of binding to the p63 30UTR (Fig. 7B).Furthermore, we found that unlike wild-type RBM24,

neither DRNP1 nor DRNP2 were capable of inhibitingDNp63a expression in HaCaT cells (Fig. 7C). Takentogether, these data suggest that the RNA-binding domainis required for RBM24 to bind p63 transcript and conse-quently, inhibits p63 expression.

DiscussionAlthough regulation of p63 expression has been exten-

sively studied, very little is known about the posttranscrip-tional regulation of p63 by either RBPs or miRNAs. RBPsare key regulators in posttranscriptional control of RNAs andaltered expression of RBPs is implicated in several kinds ofhuman diseases including cancer (33–36). In this study, weidentified RNA-binding protein RBM24 as a novel regulatorof p63 via mRNA stability. Specifically, we showed that thelevels of p63 protein and transcript are decreased by ectopicexpression of RBM24. Consistent with this, knockdown ofendogenous RBM24 increases the levels of p63 transcriptand protein. Moreover, we showed that RBM24 inhibitedp63 expression by reducing the half-life of p63 transcript.Consistently, RBM24 is able to bind to multiple regions inthe 30UTR of p63 transcript and subsequently, destabilizep63 transcript. Furthermore, we showed that both the30UTR in p63 transcript and the RNA-binding domain inRBM24 are required for regulating p63 expression.The biological function of RBM24 and its downstream

targets remain largely unknown. To date, RBM24 is sug-gested to be involved in skeletal muscle differentiation byregulating MyoD (37) and myogenin (38). More recently,RBM24 is found to be involved in sarcomeric assembly andcardiac contractility (39, 40), suggesting a critical role ofRBM24 in heart development. However, as a RBM24paralogue, RBM38 was found to be critical in tumorigenesis(41, 42). Therefore, it is likely that RBM24 and RBM38have their own distinct functions, although both proteinsshare high degree of sequence similarity. In our study, wefound that p63 is a novel downstream target of RBM24.Although the biologic significance of this regulation remainsunknown, it is likely that RBM24 participates in the p63network by regulating p63 expression via various pathways.For example, RBM24 may play a role in TAp63-mediatedtumor suppression or in DNp63-mediated epidermal devel-opment. Moreover, RBM38 is a target of the p53 family andforms a feedback or feed-forward regulatory loop with thep53 family proteins (18, 29, 43, 44). As a RBM38 closelyrelated protein, RBM24 may participate in the p53 family-RBM38 autoregulatory loop, including regulation of p53and p73 expression. Thus, future studies to address thesequestions will help us better understand the biologicalfunction of RBM24.We have previously reported that RBM38 is able to

regulate p63 mRNA stability by binding to the 30UTR ofp63 transcript (18). In this study, we found that likeRBM38, RBM24 was able to bind to the p63 30UTR anddestabilize p63 transcript. Of note, the binding sites ofRBM24 to p63 transcripts are located in the same regionsas that of RBM38 (Fig. 6 and ref. 18). We postulate that the

A

B C

RB

M24

Ctr

l3′UTR-C

1 2 3 4

1 2 3 4

1.0 0.5 1.0 1.1

RBM24

RBM24

ΔRNP2

ΔRNP1

ΔRNP2ΔRNP1

ΔRN

P2

ΔRN

P1

RNP2

HaCaT

RNP2

1 13 18 52 59 236

RNP1

RNP1

GST

RPC

RBM24

Actin

ΔNp63α

Figure 7. The RNA-binding domain is required for RBM24 to inhibit p63expression. A, schematic representation of wild-type RBM24, DRNP1,and DRNP2. B, neither DRNP1 nor DRNP2 is able to bind to the p6330UTR. REMSA was performed by incubating 32P-labeled probe C withrecombinant GST or GST-tagged RBM24, DRNP1, or DRNP2. Thebracket indicates the RNA–protein complexes. C, the RNA-bindingdomain is required for RBM24 to inhibit p63expression.HaCaTcellsweretransiently transfected with a control vector or a vector expressingRBM24,DRNP1, orDRNP2 for 48 hours. The relative level ofDNp63awasnormalized to that of actin and arbitrary set as 1.0 in control cells. Therelative fold changes were shown below each lane.






similar regulation to p63 by RBM38 and RBM24 is becauseof their high degree of sequence similarity (Fig. 1A). Inter-estingly, RBM24 can regulate p63 expression in the absenceof RBM38 (Figs. 3F and 4C), suggesting that the function ofthese 2 proteins may not be redundant. Nevertheless, it stillremains to be elucidated whether RBM24 and RBM38cooperatively or antagonistically regulate p63 mRNA sta-bility. First, RBM24 and RBM38 may compete to bind top63 transcript. Second, it is likely that RBM24 enhances theRNA-binding activity of RBM38 to p63 transcript and viceversa, resulting in destabilized p63 transcript. Third, becauseof their high degree of sequence similarity, RBM24 andRBM38 may form a heterodimer and negatively regulatep63 mRNA stability. These issues need to be addressed inthe future studies.Of note, severalmiRNAs, includingmiR-130b (21),miR-

302 (20), and miR-203 (22), are found to posttranscrip-tionally regulate p63 expression. In addition, it is now wellaccepted that RBPs work closely with miRNA to eitherpositively or negatively modulate their target expression. Insupport of this idea, RBM38 was found to modulate theability of several miRNAs to bind to their targets (45).Therefore, it will be interesting to determine whetherRBM24 alone or together with RBM38 is able to modulatethe ability of miRNAs to bind to p63 transcripts andsubsequently, affect p63 activity. By addressing these ques-

tions, it will help us further understand how posttranscrip-tional regulatory mechanisms contribute to p63 expressionand consequently, affect the p63 network.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: E. Xu, J. Zhang, X. ChenDevelopment of methodology: E. Xu, J. Zhang, M. Zhang, Y. Jiang, S.-J. ChoAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): X. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): E. Xu, J. Zhang, X. ChenWriting, review, and/or revision of the manuscript: E. Xu, J. Zhang, X. ChenAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): E. Xu, Y. Jiang, S.-J. ChoStudy supervision: J. Zhang, X. Chen

AcknowledgmentsThe authors thank Dr. E.R. Flores for providing the TAp63 knockout mice.

Grant SupportThis work is supported in part by an NIH grant (CA102188).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 3, 2013; revised November 22, 2013; accepted December 11,2013; published OnlineFirst December 27, 2013.

References1. YangA, KaghadM,WangY,Gillett E, FlemingMD,Dotsch V, et al. p63,

a p53 homolog at 3q27-29, encodes multiple products with transacti-vating, death-inducing, and dominant-negative activities. Mol Cell1998;2:305–16.

2. Harms K, Nozell S, Chen X. The common and distinct target genes ofthe p53 family transcription factors. Cell Mol Life Sci 2004;61:822–42.

3. MangiulliM, Valletti A,CaratozzoloMF, Tullo A, Sbisa E, PesoleG, et al.Identification and functional characterization of two new transcription-al variants of the human p63 gene. Nucleic Acids Res 2009;37:6092–104.

4. Vanbokhoven H, Melino G, Candi E, Declercq W. p63, a story of miceand men. J Invest Dermatol 2011;131:1196–207.

5. Dohn M, Zhang S, Chen X. p63a and DNp63a can induce cell cyclearrest and apoptosis and differentially regulate p53 target genes.Oncogene 2001;20:3193–205.

6. Helton ES, Zhu J, Chen X. The unique NH2-terminally deleted (DN)residues, the PXXP motif, and the PPXY motif are required for thetranscriptional activity of the DN variant of p63. J Biol Chem2006;281:2533–42.

7. Candi E, Dinsdale D, Rufini A, Salomoni P, Knight RA, Mueller M, et al.TAp63 and DNp63 in cancer and epidermal development. Cell Cycle2007;6:274–85.

8. Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, Hill DE, et al.AIS is an oncogene amplified in squamous cell carcinoma. Proc NatlAcad Sci U S A 2000;97:5462–7.

9. Su X, Chakravarti D, Cho MS, Liu L, Gi YJ, Lin YL, et al. TAp63suppresses metastasis through coordinate regulation of Dicer andmiRNAs. Nature 2010;467:986–90.

10. Mills AA, ZhengB,WangXJ, Vogel H, RoopDR, Bradley A. p63 is a p53homologue required for limb and epidermal morphogenesis. Nature1999;398:708–13.

11. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, et al.p63 is essential for regenerative proliferation in limb, craniofacial andepithelial development. Nature 1999;398:714–8.

12. Romano RA, Smalley K, Magraw C, Serna VA, Kurita T, Raghavan S,et al. DNp63 knockout mice reveal its indispensable role as a masterregulator of epithelial development and differentiation. Development2012;139:772–82.

13. Su X, Gi YJ, Chakravarti D, Chan IL, Zhang A, Xia X, et al. TAp63 is amaster transcriptional regulator of lipid and glucose metabolism. CellMetab 2012;16:511–25.

14. Su X, Paris M, Gi YJ, Tsai KY, Cho MS, Lin YL, et al. TAp63 preventspremature aging by promoting adult stem cell maintenance. Cell StemCell 2009;5:64–75.

15. Harmes DC, Bresnick E, Lubin EA, Watson JK, Heim KE, Curtin JC,et al. Positive and negative regulation of DN-p63 promoter activity byp53 and DN-p63-a contributes to differential regulation of p53 targetgenes. Oncogene 2003;22:7607–16.

16. Qian Y, Jung YS, Chen X. DNp63, a target of DEC1 and histonedeacetylase 2,modulates the efficacyof histonedeacetylase inhibitorsin growth suppression and keratinocyte differentiation. J Biol Chem2011;286:12033–41.

17. Herfs M, Hubert P, Suarez-Carmona M, Reschner A, Saussez S, BerxG, et al. Regulation of p63 isoforms by snail and slug transcriptionfactors in human squamous cell carcinoma. Am J Pathol 2010;176:1941–9.

18. Zhang J, Jun Cho S, Chen X. RNPC1, an RNA-binding protein and atarget of the p53 family, regulates p63 expression through mRNAstability. Proc Natl Acad Sci U S A 2010;107:9614–9.

19. Yan W, Zhang Y, Zhang J, Cho SJ, Chen X. HuR is necessary formammary epithelial cell proliferation and polarity at least in part viaDNp63. PLoS One 2012;7:e45336.

20. Scheel AH, Beyer U, Agami R, Dobbelstein M. Immunofluorescence-based screening identifies germ cell associated microRNA 302 as anantagonist to p63 expression. Cell Cycle 2009;8:1426–32.

21. Rivetti di Val Cervo P, Lena AM, NicolosoM, Rossi S, Mancini M, ZhouH, et al. p63-microRNA feedback in keratinocyte senescence. ProcNatl Acad Sci U S A 2012;109:1133–8.

Xu et al.





22. Lena AM, Shalom-Feuerstein R, Rivetti di Val Cervo P, Aberdam D,Knight RA, Melino G, et al. miR-203 represses 'stemness' by repres-sing DNp63. Cell Death Differ 2008;15:1187–95.

23. Rossi M, Aqeilan RI, Neale M, Candi E, Salomoni P, Knight RA, et al.The E3 ubiquitin ligase Itch controls the protein stability of p63. ProcNatl Acad Sci U S A 2006;103:12753–8.

24. Jung YS, Qian Y, Yan W, Chen X. Pirh2 E3 ubiquitin ligase modulateskeratinocyte differentiation through p63. J Invest Dermatol 2013;133:1178–87.

25. Li Y, Zhou Z, Chen C. WW domain-containing E3 ubiquitin proteinligase 1 targets p63 transcription factor for ubiquitin-mediated pro-teasomal degradation and regulates apoptosis. Cell Death Differ2008;15:1941–51.

26. Gallegos JR, Litersky J, Lee H, Sun Y, Nakayama K, Lu H. SCF TrCP1activates and ubiquitylates TAp63g. J Biol Chem 2008;283:66–75.

27. Yan W, Chen X, Zhang Y, Zhang J, Jung YS. Arsenic suppresses cellsurvival via Pirh2-mediated proteasomal degradation of DNp63 pro-tein. J Biol Chem 2013;288:2907–13.

28. Harms KL, Chen X. Histone deacetylase 2 modulates p53 transcrip-tional activities through regulation of p53-DNAbinding activity. CancerRes 2007;67:3145–52.

29. Zhang J, Cho SJ, Shu L, YanW,Guerrero T, KentM, et al. Translationalrepression of p53 by RNPC1, a p53 target overexpressed in lympho-mas. Genes Dev 2011;25:1528–43.

30. Scoumanne A, Cho SJ, Zhang J, Chen X. The cyclin-dependent kinaseinhibitor p21 is regulated by RNA-binding protein PCBP4 via mRNAstability. Nucleic Acids Res 2011;39:213–24.

31. Cho SJ, Zhang J, Chen X. RNPC1modulates the RNA-binding activityof, and cooperates with, HuR to regulate p21 mRNA stability. NucleicAcids Res 2010;38:2256–67.

32. Peritz T, Zeng F, Kannanayakal TJ, Kilk K, Eiriksdottir E, Langel U, et al.Immunoprecipitation ofmRNA-protein complexes. Nat Protoc 2006;1:577–80.

33. Heinonen M, Bono P, Narko K, Chang SH, Lundin J, Joensuu H, et al.Cytoplasmic HuR expression is a prognostic factor in invasive ductalbreast carcinoma. Cancer Res 2005;65:2157–61.

34. Denkert C, Koch I, von Keyserlingk N, Noske A, Niesporek S, Dietel M,et al. Expression of the ELAV-like protein HuR in human colon cancer:

association with tumor stage and cyclooxygenase-2. Mod Pathol2006;19:1261–9.

35. Chen AJ, Paik JH, Zhang H, Shukla SA,Mortensen R, Hu J, et al. STARRNA-binding protein quaking suppresses cancer via stabilization ofspecific miRNA. Genes Dev 2012;26:1459–72.

36. Wang XY, Penalva LO, Yuan H, Linnoila RI, Lu J, Okano H, et al.Musashi1 regulates breast tumor cell proliferation and is a prognosticindicator of poor survival. Mol Cancer 2010;9:221.

37. Li HY, Bourdelas A, Carron C, Shi DL. The RNA-binding proteinSeb4/RBM24 is a direct target of MyoD and is required for myogen-esis during Xenopus early development. Mech Dev 2010;127:281–91.

38. Jin D, Hidaka K, Shirai M, Morisaki T. RNA-binding motif protein 24regulates myogenin expression and promotes myogenic differentia-tion. Genes Cells 2010;15:1158–67.

39. Poon KL, Tan KT, Wei YY, Ng CP, Colman A, Korzh V, et al. RNA-binding protein RBM24 is required for sarcomere assembly and heartcontractility. Cardiovasc Res 2012;94:418–27.

40. Maragh S, Miller RA, Bessling SL, McGaughey DM, Wessels MW, deGraaf B, et al. Identification of RNA binding motif proteins essential forcardiovascular development. BMC Dev Biol 2011;11:62.

41. Hermsen M, Postma C, Baak J, Weiss M, Rapallo A, Sciutto A, et al.Colorectal adenoma to carcinoma progression follows multiplepathways of chromosomal instability. Gastroenterology 2002;123:1109–19.

42. Carvalho B, Postma C, Mongera S, Hopmans E, Diskin S, van de WielMA, et al. Multiple putative oncogenes at the chromosome 20qamplicon contribute to colorectal adenoma to carcinoma progression.Gut 2009;58:79–89.

43. Shu L, YanW, Chen X. RNPC1, an RNA-binding protein and a target ofthe p53 family, is required for maintaining the stability of the basal andstress-induced p21 transcript. Genes Dev 2006;20:2961–72.

44. Yan W, Zhang J, Zhang Y, Jung YS, Chen X. p73 expression isregulated by RNPC1, a target of the p53 family, via mRNA stability.Mol Cell Biol 2012;32:2336–48.

45. Leveille N, Elkon R, Davalos V, Manoharan V, Hollingworth D, OudeVrielink J, et al. Selective inhibition of microRNA accessibility byRBM38 is required for p53 activity. Nat Commun 2011;2:513.






2014;12:359-369. Published OnlineFirst December 27, 2013.Mol Cancer Res Enshun Xu, Jin Zhang, Min Zhang, et al. StabilityRNA-Binding Protein RBM24 Regulates p63 Expression via mRNA

Updated version

10.1158/1541-7786.MCR-13-0526doi:

Access the most recent version of this article at:

Cited articles

http://mcr.aacrjournals.org/content/12/3/359.full#ref-list-1

This article cites 45 articles, 16 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/12/3/359.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/12/3/359To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-13-0526

http://mcr.aacrjournals.org/content/12/3/359.full#ref-list-1

http://mcr.aacrjournals.org/content/12/3/359.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/12/3/359


Molecular Cancer Research - RNA-Binding Protein RBM24 … · 2014-03-07 · Chromatin, Gene, and...

Documents

Transcript of Molecular Cancer Research - RNA-Binding Protein RBM24 … · 2014-03-07 · Chromatin, Gene, and...