Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates ...

38 Pages·2009·0.36 MB·English

Human Molecular Genetics, 2009, Vol. 18, No. 7 1229–1237
doi:10.1093/hmg/ddp023
Advance Access published on January 15, 2009
Tannic acid facilitates expression of the
polypyrimidine tract binding protein and alleviates
deleterious inclusion of CHRNA1 exon P3A due to
an hnRNP H-disrupting mutation in congenital
myasthenic syndrome
D
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Yang Bian1, Akio Masuda1, Tohru Matsuura1, Mikako Ito1, Kazuya Okushin1, n
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Andrew G. Engel2 and Kinji Ohno1,(cid:2) ad
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1Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of m
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Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan and 2Department of Neurology and Neuromuscular ttp
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Research Laboratory, Mayo Clinic, Rochester, MN, USA ://a
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ReceivedOctober10,2008;RevisedDecember5,2008;AcceptedJanuary12,2009 em
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We recently reported that the intronic splice-site mutation IVS3-8G>A of CHRNA1 that encodes the muscle .c
o
nicotinic acetylcholine receptor a subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, m
/h
resultsinexclusiveinclusionofthedownstreamexonP3A.TheP3A(1)transcriptencodesanon-functional m
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a subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional signifi- /a
cance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of rtic
le
960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to -a
b
IVS3-8G>A but without altering the expression of hnRNP H. Therefore, we searched for another splicing stra
trans-factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 30 end of c
t/1
CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of 8
/7
PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic /1
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acid-responsiveelementisbetweenpositions 2232and 274fromthetranslationinitiationsite.Theseobser- 2
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vations open the door to the discovery of novel therapies based on PTB overexpression and to detecting /67
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possible untoward effects of the overexpression. 5
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INTRODUCTION a variable number of galloyl esters. Tannins are rich in nuts, 28
red wine, tea and coffee, but hydrolyzable tannins including M
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Tannins are plant-derived polyphenols and are divided into tannic acid are not rich in tea (2,3) or red wine (4). rc
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twogroupsof hydrolyzable andcondensed tannins (proantho- Polypyrimidine tract binding protein (PTB), encoded by 2
0
cyanidins) (1). Hydrolyzable tannins are derivatives of gallic PTBP1, is multifunctional, which participates in pre-mRNA 19
acid (3,4,5-trihydroxyl benzoic acid) in which a variable splicing (5,6), internal ribosome entry site (IRES)-mediated
numberofgallicacidsareesterifiedtoacorephenol.Thesim- translation (7), mRNA polyadenylation (8), RNA localization
plesthydrolyzabletanninsaregallotanninsthatarepolygalloyl (9) and mRNA stabilization (10). PTB carries four RNA rec-
estersofglucose.Theprototypicalgallotanninistannicacidor ognition motifs and each motif binds to a CU-rich element
1,2,3,4,6-penta-O-b-D-glucose (CAS 1401-55-4, C76H52O46) (11). In terms of a splicing trans-factor, CU-rich splicing
comprised five galloyl esters and a glucose. Commercially cis-elementsarelocatedupstreamand/ordownstreamofalter-
available tannic acid additionally includes gallotannins with nativelysplicedexons.PTBboundtothe30 splicesitedirectly
(cid:2)Towhomcorrespondenceshouldbeaddressed.Tel: þ81527442446;Fax:þ81527442449;Email:[email protected]
# The Author 2009. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
1230 Human Molecular Genetics, 2009, Vol. 18, No. 7
competes with binding of U2AF65 that recognizes the poly-
pyrimidine tract (12–15). Alternatively, PTBs bound to the
flanking introns repress splicing of the exon (16–20).
We recently identified a pathogenic intronic mutation in a
congenital myasthenic syndrome, IVS3-8G.A of CHRNA1
that encodes the muscle nicotinic acetylcholine receptor a
subunit (21). We reported that IVS3-8G.A disrupts binding
of a splicing repressor, hnRNP H, and causes exclusive
inclusionofthedownstreamexonP3A.TheP3A(þ)transcript
encodesanon-functionalasubunitandcomprises50%ofthe
transcriptsinnormalhumanskeletalmuscle,butitsfunctional Figure1.ChimericCHRNA1exonP3Aminigeneforscreeningofchemical
compounds.CHRNA1exonP3Aanditsflankingintronsareinsertedafterpos-
significance remains unknown (22–24).
ition834(arrowhead)ofthefireflyluciferasecDNA.SkippingofexonP3A D
Here, we report that PTB binds to the polypyrimidine tract should generate an intact luciferase molecule, whereas inclusion of exon ow
of CHRNA1 intron 3 immediately upstream of the hnRNP P3Ashouldabrogateit.ThehnRNPH-binding‘TGGG’motifisunderlined. nlo
H-binding site, and silences recognition of exon P3A in ad
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cooperation with hnRNP H. IVS3-8G.A, however, has no d
effect on the binding of PTB. Screening of chemical com- Table1. Twenty-fourbestcompoundswithaveragednormalizedrelative from
pounds revealed thattannicacid mitigates exclusive inclusion luciferaseactivity(cid:3)1.20andCV%,0.20(n¼4) h
of exon P3A due to IVS3-8G.A by activating the promoter ttp
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region of PTBP1 and causes a dose-dependent increase of Compounds Mean SD ://a
PTBP1 mRNA. ca
Tomatine 1.50 0.16 d
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Tannicacid 1.47 0.20 m
Troxerutin 1.44 0.22 ic.o
RESULTS Camptothecin 1.43 0.12 up
Mexiletinehydrochloride 1.43 0.21 .c
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Screening of off-label effects of 960 chemical compounds Halcinonide 1.42 0.07 m
Clobetasolpropionate 1.40 0.21 /h
In an effort to search for a potential therapeutic modality 6a-Methylprednisoloneacetate 1.40 0.27 m
g
for aberrant inclusion of CHRNA1 exon P3A due to Flurandrenolide 1.33 0.19 /a
IVS3-8G.A, we screened for off-label effects of 960 chemi- Oxethazaine 1.31 0.11 rtic
Ketorolactromethamine 1.28 0.09 le
cal compounds (GenPlus, MicroSource Discovery Systems). Avocadynoneacetate 1.27 0.10 -ab
Mcoomsptowunerdes FoDr Ana-atuprparlovperoddudcrutsg.s,Waendcosnosmtruectweedrea bcihoiamcetirvice MPreodprayfseonnoenehydrochloride 11..2266 00..1109 strac
minigene carrying the mutant exon P3A with its flanking Etodolac 1.24 0.11 t/1
8
introns in the middle of the firefly luciferase cDNA Propylthiouracil 1.23 0.06 /7
Pergolidemesylate 1.23 0.03 /1
(pcDNA3.1-Luc-mtP3A) (Fig. 1). Skipping of exon P3A 2
Hydrocortisonebutyrate 1.22 0.10 2
should generate luminescence, whereas its inclusion should 5-Azacytidine 1.22 0.05 9/6
not. After the first round of screening in duplicate, we nar- Alexidinehydrochloride 1.22 0.10 73
rowed the list to 80 compounds and performed the second Fluconazole 1.21 0.10 53
round of screening in duplicate. Among the 80 compounds, Methoxyaminehydrochloride 1.21 0.09 3 b
N-Formylmethionylalanine 1.21 0.11 y
24 consistently exhibited beneficial effects (Table 1). We Dinitolmide 1.20 0.08 gu
observed no shared feature among the 24 compounds. We es
hereafter focused on tannic acid, because tannic acid was Amongthe80compoundsthatweemployedforthesecondroundof t on
expected to have less toxicity or untoward effects than the screening,24compoundsconsistentlydemonstratedbeneficialresponses. 2
8
other compounds. ThevaluesaretheglobalmeanandSDofthefirst(n¼2)andsecond M
(n¼2)roundsofscreening. a
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0
Tannic acid induces skipping of CHRNA1 exon P3A 19
Tannic acid has no effect on the expression of hnRNP H
in a dose-dependent manner
Wenextexaminedadose-dependenteffectoftannicacid.We We next searched for the molecular basis of the tannic acid-
added 0, 1, 10 and 100mM of tannic acid to transfected mediated amelioration of the aberrant inclusion of exon
HEK293T cells and examined its effect on splicing of exon P3A. We previously reported that recognition of exon P3A
P3A by real-time RT–PCR. Tannic acid exhibited no effect isdown-regulatedbybindingofhnRNPHto‘UGGG’atpos-
on splicing of wild-type exon P3A, whereas 100mM tannic itions 211 to 28 of intron 3 (Fig. 1), and IVS3-8G.A
acid minimally but significantly increased the ratio of the attenuates its bindings (cid:2)100-fold (21). We hypothesized
P3A(2)-transcript(Fig.2AandB).Lackofaneffectonwild- thattannicacidincreasestheexpressionofhnRNPHandsub-
type exon P3A may represent that the wild-type exon P3A is sequently silences the recognition of exon P3A. We therefore
mostly spliced out even in the absence of tannic acid and analyzed the effect of tannic acid on mRNA levels of hnRNP
thatwecannotobserveaneffectoftannicacidinthewild-type H in HEK293T cells by real-time RT–PCR, but found no
construct. effect (data not shown).
Human Molecular Genetics, 2009, Vol. 18, No. 7 1231
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Figure2.TannicacidalleviatesaberrantinclusionofCHRNA1exonP3Adue trac
to IVS3-8G.A by facilitating the expression of PTBP1. (A) RT–PCR of t/1
wild-typeandmutantCHRNA1minigenesinHEK293Tcellswithincreasing 8/7
concentrationsoftannicacid.(B)Real-timeRT–PCRtoquantifytheratioof /1
P3A-skippedtranscriptarisingfromwild-typeandmutantCHRNA1minigenes 22
inHEK293Tcellswithincreasingconcentrationsoftannicacid.Theratiosare 9/6
representedbythemeanandSD.Forthemutantminigene,100mMtannicacid 73
increases the ratio of the P3A(2)-transcript. (C) Real-time RT–PCR to 5
demonstrate a dose-dependent increase of PTBP1 mRNA by tannic acid. Figure3.PTBbindsclosetothe30splicesiteofexonP3Aandinducesskip- 33
The ratios are normalized to that in the absence of tannic acid. Means and ping of exon P3A. (A) 32P-labeled RNA probe is incubated with nuclear by
SDarerepresented. extracts of indicated cells, followed by UV cross-linking and SDS–PAGE. g
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Autoradiographs demonstrate a (cid:2)60-kDa molecule (arrowhead). (B) e
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32P-labeledRNAprobeisincubatedwithnuclearextractsofHEK293Tcells t o
followed by UV cross-linking and RNase digestion. The bound molecules n
PTB binds to CHRNA1 intron 3 and silences splicing 2
are immunoprecipitated with anti-PTB (At-PTB) or control antibody (cont 8
of exon P3A Ab), and separated on SDS–PAGE. Only anti-PTB antibody precipitates a M
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(cid:2)60-kDamolecule.(C)Westernblottingwithanti-PTBoranti-SRp55anti- rc
We next looked for a new splicing trans-factor that regulates bodyofaffinitypurifiedHEK293Tnuclearextract.Affinitypurificationalso h
2
recognitionofexonP3A.UVcross-linkingofnuclearextracts demonstratesthebindingofPTBtothewild-type(WT)intron3,butnotto 0
1
of HeLa, HEK293T and C2C12 cells to wild-type CHRNA1 thescrambledRNAprobe(Scr).NUCrepresentsalaneloadedwithnascent 9
nuclear extract. (D) Real-time RT–PCR analysis of HeLa cells transfected
intron3disclosed a (cid:2)60kDa fragment (Fig.3A).Itsmolecu-
withthewild-typeminigenealongwiththeindicatedsiRNAorcDNA.The
larweightpromptedustohypothesizethattheboundmolecule %P3Avaluesarenormalizedtothoseofcontrolvalues.Down-regulationof
was PTB. Immunoprecipitation of the UV-cross-linked PTBP1enhances,whereasoverexpressionofPTBP1silencestherecognition
nuclear extract (Fig. 3B) and western blotting of affinity ofexonP3A.BarsrepresentthemeanandSDofthreeexperiments.
purified molecules (Fig. 3C) indeed identified that the bound
molecule was PTB. Additionally, siRNA-mediated down- shown). As we previously found that hnRNP H binds close
regulation of PTB enhanced recognition of exon P3A, to the 30 end of intron 3 and silences splicing of exon P3A
whereas overexpression of PTB silenced it (Fig. 3D), which (21), we asked if hnRNP H and PTB interact with each
supports the notion that PTB binds to intron 3 and induces other. Anti-hnRNP H antibody did not co-immunoprecipitate
skipping of exon P3A. PTB in a nuclear extract of HEK293T cells in the absence
Surface plasmon resonance analysis demonstrated that of substrate mRNA (data not shown). To summarize, splicing
IVS3-8G.A had no effect on the binding of PTB (data not of exon P3A is down-regulated by both hnRNP H and PTB,
1232 Human Molecular Genetics, 2009, Vol. 18, No. 7
reduced the responses to tannic acid but not to the level of
the74-nucleotideconstruct.Real-timeRT–PCRalsorevealed
that 1, 10 and 100mM of tannic acid did not increase the
expression level of the SP1 gene encoding Sp1 or the
TFAP2AgeneencodingAP-2a(datanotshown).Tosummar-
ize, tannic acid-responsive cis-element likely resides between
positions 232 and 74 of PTBP1, but the exact transcriptional
cis-element remains unidentified.
DISCUSSION
PTB silences splicing of exon P3A D
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WeidentifiedthatPTBbindsclosetothe30endofintron3and n
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silencessplicingofthedownstreamexonP3A.Inourprevious a
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studies, Syproruby staining of the affinity purified nuclear ed
extract disclosed binding of hnRNP H but not of PTB (21). fro
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In our current studies, UV cross-linking of the nuclear h
extract to a radiolabeled probe detected binding of PTB but ttp
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not of hnRNP H. Binding of hnRNP H and PTB, as well as ://a
their silencing effects on splicing of exon P3A, have been c
a
confirmed by several other methods for both factors. For de
m
RNA-binding molecules, we frequently observe this kind of ic
Figure4.ScanningdeletionanalysisofthePTBP1promoterregion.(A)Luci- inconsistency. This is likely because an RNA-binding .o
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fluercaifseerapsreomaoctteivriatsiessaysarteodfiermstoncsatlrcautelarteesdpobnysedsitvoid8i0ngmMthtaennfiirceflacyidl.uRceifleartaivsee molecule recognizes the tertiary structure of RNA (25,26), p.co
activitybythecotransfectedRenillaluciferaseactivity.Thecalculatedrelative and the tertiary structure likely differs depending on the m
luciferaseactivityinthepresenceof80mMtannicacidisthendividedbythat size, position and the attached molecule of the RNA probe /hm
intheabsenceoftannicacidtoestimatetheresponse.MeansandSD(n¼4) employed for the binding assay. g
ainreitiraetpiorensseinteteadr.eTihnedisciazteesdo.f‘2th3e2-PSTpB1’Pa1npdr‘o2m3o2t-eArPr2eg’iionndibceafteorae2th3e2-tnraunclselaottiiodne AlthoughbothPTBandhnRNPHbindtothesimilarintro- /artic
nicregionandbothsilencesplicingofexonP3A,wefailedto le
constructlackingtwoSp1elements[underlinedin(B)]orthreeAP2elements -a
[indicated by dots in (B)], respectively. (B) PTBP1 promoter region from detect a direct interaction of the two molecules. Markovtsov b
s
position –239to þ3.Arrowheadspoint tothe 50 endsofthe 232-nucleotide et al. (27) similarly found that hnRNP H/F and PTB/nPTB tra
and74-nucleotideconstructsin(A). cooperatively enhance binding to a splicing cis-element of ct/1
the c-src N1 exon, but did not demonstrate a direct protein– 8
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and IVS3-8G.A only compromises binding of hnRNP H but protein interaction. Further analysis is required to prove if /1
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not of PTB. thetwo molecules silence splicing of exon P3Acooperatively 2
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or independently. /6
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3
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TannicacidinducestheexpressionofPTBbyactivatingits 33
promoter region Off-label effects of FDA-approved drugs by
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We next asked if tannic acid exerts its effect by inducing FDA-approved drugs and bioactive compounds have been ue
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expression of PTB, and found that tannic acid increased the employed for screening of off-label effects for neurological t o
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expression of PTBP1 mRNA in a dose-dependent manner diseases, because the identified compounds can be readily 2
(Fig. 2C). We further asked how PTBP1 is transcriptionally applied to clinical practice (28,29). The identified off-label 8 M
induced by tannic acid, and examined the effect of tannic compoundsincludedigitoxin,nerifolin,peruvosideandsuloc- a
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acidonthePTBP1promoterregionusingtheluciferaserepor- tidil for spinobulbar muscular atrophy (30); b-lactam anti- h
2
ter assay (Fig. 4). We measured responses to 80mM of tannic biotics for amyotrophic lateral sclerosis and brain ischemia 01
acid in constructs harboring 1626, 1187, 830, 711, 295, 232 (31); and dorsomorphin for fibrodysplasia ossificans progres- 9
and 74 nucleotides upstream of the translation initiation site siva (32). Additionally, kinetin alleviates aberrant splicing of
of the human PTBP1 gene. Tannic acid increases luciferase IKBKAP in familial dysautonomia (33). (2)-Epigallocatechin
activity (cid:2)2.5-fold when the promoter length is 711 nucleo- gallate also down-regulates the expression of hnRNP A2/B1
tides or longer (Fig. 4A). This degree of response is similar and normalizes the aberrant splicing of mutant IKBKAP
to that observed with the native PTBP1 gene (Fig. 2C). The (34). Similarly, sodium valproate corrects splicing of SMN2
295- and 232-nucleotide constructs also respond to tannic by increasing the expression of a splicing trans-factor,
acidbut to alesser extent.The74-nucleotide construct exhib- Htra2-b1, and also increases the expression of SMN2
ited no response to tannic acid. We then asked why the (35,36). Sodium valproate is a histone deacetylase (HDAC)
232-nucleotide construct responds to tannic acid, whereas inhibitor and activates a variety of genes (37). We identified
the74-nucleotide constructdoesnot.We deletedtwoputative that tannic acid works on the promoter region of PTBP1 and
Sp1sitesandthreeputativeAP2sitesfromthe232-nucleotide facilitates its expression in a dose-dependent manner. Tannic
construct (Fig. 4B). Both deletion constructs minimally acid, however, is unlikely to act directly on the promoter
Human Molecular Genetics, 2009, Vol. 18, No. 7 1233
region of PTBP1, but rather modulates unidentified cellular 60% body water in a 60kg human, the tannic acid concen-
processes that culminate in the activation of the PTBP1 pro- trations in body water would become 8–16mM. Our studies
moter region. The exact activation mechanisms still remain using HEK293T cells demonstrate that even 1mM of tannic
to be elucidated. acid up-regulates the expression of PTBP1 but 100mM of
tannic acid is required to attenuate the aberrant splicing of
exon P3A. As the absorption efficiency and elimination rate
Therapeutic effects of tannic acid in other diseases
of dietary tannic acid has not been determined in humans,
Tannic acid has protein-aggregating, astringent, anti- we cannot predict how much tannic acid would be required
microbial, anti-oxidant, anti-mutagenic and anti-proliferative to treat patients, and whether it is toxic or not.
properties (38). For example, the constringing action of
tannic acid on mucous tissues is exploited to treat diarrhea
in medical practice. Tannic acid inhibits the 3C-like protease Diseases and physiological states in which the D
encoded by the severe acute respiratory syndrome (SARS) up-regulation of PTB is beneficial or detrimental ow
coronavirus with IC50 of 3mM (39). Tannic acid has anti- nlo
oxidantproperties(40)andisprotectiveagainsttoxin-induced Raponi et al. (54) recently reported that PTB recognizes two ad
splicing cis-elements in the pseudoexon of NF1 activated by e
skin tumors (41). Tannic acid similarly suppresses skin tumor d
promotion induced by UV-B radiation by 70% in mice (42). an A-to-G mutation 270 nucleotides upstream of the nascent fro
intron/exon boundary. Up-regulation of PTB ameliorated the m
Tannic acid is protective against spontaneous liver tumor h
development in mice by 87% (43). In addition, tannic acid aberrant inclusion of the pseudoexon. Fred and Welsh (10) ttp
r3a0i%ses(t4h4e).surAvpivoapltorsaitse-ionfdumciicnegbaecatriivnigtysyonfgentaenicnictumaocirds biys fboinudnidngthtoatitsPT30B-UTsRtabainlidzepsartthiceipaptreepinrotihnesuglilnucomseR-NinAducbedy s://aca
increase of preproinsulin mRNA. In both of these conditions, d
mediated by suppression of protease activity in tumor cells e
up-regulation of PTB by tannic acid might be beneficial. m
(45),reductionofERK1,2phosphorylationbyacyclicadeno- ic
sine 50,30-monophosphate-protein kinase A-dependent Ontheotherhand,PTBisoverexpressedintumorcellsand .ou
promotes cell growth (55,56), although PTB itself does not p
pathway (46) and also down-regulation of the superoxide dis- .c
mutase (47). Up-regulation of PTB by tannic acid, however, transform cells (57). Viruses exploit PTB to facilitate the om
has never been reported to our knowledge. IRES-mediated translation initiation (58,59). PTB also /hm
silences the splicing of a cassette exon of the neuronal PTB g
/a
Potential clinical application of tannic acid (nnePuTroBn)alancedllds.owInn-nreeugruolnaatelsctehlles,emxpircersosRioNnAofmniRP-T1B24indonwonn-- rticle
The LD of orally administered tannic acid is as high as regulates the translation of PTB and hence up-regulates the -ab
50 s
52020600mmgg//kkgg (i4n9).raTthe(48h)u.mRanabtbhirteshhoasld,athehigtohleerrabLlDe5d0aiolyf eoxfpPrTesBsiobny toafnnniPcTaBcid(6c0o)u.lIdntothbeseedceotrnimdietinotnasl., up-regulation tract/1
Although tannic acid in the form of albumin tannate has 8
intake (TDI), is extrapolated from the rodent threshold, the /7
no observed adverse effect level (NOAEL), by dividing the long been used as a safe anti-diarrhea medication, our obser- /1
2
NOAEL with the uncertainty factor (UF) (50). Assuming vations that tannic acid causes overexpression of PTB now 29
that the default UF representing the interspecies variation is opens the door to the discovery of beneficial as well as poss- /67
ible detrimental effects of PTB overexpression. 3
10 (51,52), the NOAELs of 2260 and 5000mg/kg should 5
3
give rise to the TDIs of 13.6 and 30.0g of tannic acid for a 3 b
60kg human, respectively. If these doses were completely y g
absorbed and evenly dissolved in 60% body water, the MATERIALS AND METHODS ue
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taanndTnhi4ce90aadcmivdMercsionenehcfuefnmectratasnt.ioofntsaninnicboadcyidwhaavteernwootubledenbethcoormoeug2h2ly1 FexiroenflPy3lAuciferase minigene carrying chimeric CHRNA1 t on 28 M
investigatedinhumans.Koideetal.(44)reportthat8750mg/ We screened for off-label effects of 960 chemical compounds a
rc
kg/day of dietary tannic acid for 35 days reduces the weight using chimeric minigenes carrying the firefly luciferase cDNA h
2
gain in mice, but causes no pathological changes in kidney, (Fig. 1). We first inserted the luciferase gene derived from the 0
1
liver or lung. This would be equivalent to 52.5g/day for a pGL3-Basicvector(Promega)intotheCMV-basedmammalian 9
60kg human according to the NOAEL approach described expressionvector,pcDNA3.1(Invitrogen),tomakepcDNA3.1-
above. Afsana et al. (53) report that 20g/kg/day of dietary Luc. Using the megaprimer-based site-directed mutagenesis
tannic acid for 3 weeks has no effect on the body weight method (61), we inserted a chimeric intron of 50-GTAAGTA
gain or food intake in rats, but 10g/kg/day or more of TCAAGCGGCCGCNNNNNTTAATTAATCTTACTGACAT
tannic acid for 3 weeks induces anemia by reducing the iron CCACTTTGCCTTTCTCTCCACAG-30 at position 834 of the
absorption, which would be equivalent to 60g/day for a luciferase cDNA, where position þ1 represents the translation
60kg human according to the NOAEL approach. initiation site. The underlined are NotI and PacI restriction
Clinically available tannic acid is albumin tannate (CAS sites. We next inserted CHRNA1 exon P3A with its flanking
9006-52-4) composed of 50% tannic acid and 50% albumin. intronsof150and90nucleotidesusingtheNotIandPacIsites
Inclinicalpractice,1–2gofalbumintannateisgivenfordiar- to make pcDNA3.1-Luc-wtP3A. With this construct, the
rhea which is equivalent to 0.5–1g of tannic acid. If these nonsense-mediated mRNA decay is not provoked even when
doses were completely absorbed and evenly dissolved in exon P3A is spliced in. We then engineered IVS3-8G.A
1234 Human Molecular Genetics, 2009, Vol. 18, No. 7
usingtheQuikChangesite-directedmutagenesiskit(Stratagene), P3A(2)-transcripts by real-time RT–PCR using Mx3000P
andmadepcDNA3.1-Luc-mtP3A. (Stratagene) as previously described (21). Each real-time
For all clones, the presence of the desired mutation and the RT–PCR experiment was performed in triplicate. Patient
absence of artifacts was determined by sequencing the entire cellswereunavailablefortheanalysisofeffectsoftannicacid.
insert with the CEQ8000 DNA analysis system (Beckman
Coulter).
In vitro UV cross-linking and immunoprecipitation to
isolate an RNA-binding protein
Screening of off-label effects of 960 chemical compounds
We synthesized [a-32P]-CTP labeled RNA using the Ribop-
HEK293T cells were maintained in the Dulbecco’s modified robe System (Promega) from a PCR-amplified fragment
Eagle’s medium supplemented with 10% fetal calf according to the manufacturer’s instructions. The forward
serum (Sigma-Aldrich). At (cid:2)50% confluency in a 75cm2 primer was 50-TAATACGACTCACTATAGGGAGACAGG-30,
D
flask, we transfected 6mg of either pcDNA3.1-Luc or where the italicized is T7 promoter and the underlined is for o
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pcDNA3.1-Luc-mtP3A along with 2mg of phRL-SV40 annealing to the reverse primer. The reverse primer was nlo
encoding the Renilla luciferase (Promega) using 24ml of the 50-CATGTCACCCTGTCCACCCACAGAAAAGGAGCCTG ad
e
FuGENE6 transfection reagent (Roche). Sixteen hours after TCTC-30. Nuclear extracts were prepared as previously d
transfection, we removed the transfection reagent and split described (62). The radioactively labeled RNA (1(cid:4) fro
m
the cells into three 96-well plates containing 10mM of each 104cpm) was incubated at room temperature with 2ml of h
chemical compound (GenPlus) and 2% DMSO. After nuclear extracts, 8mg of yeast tRNA and 0.8U of RNasin ttp
s
growing the cells for 24h, we measured the firefly and (Toyobo) in a final volume of 10ml of the binding buffer ://a
Renilla luciferase activities with the Dual luciferase reporter (20mM HEPES pH 7.8, 50mM KCl, 3mM MgCl2, 0.5mM cad
assay system (Promega) using the Luminoskan Ascent lumin- dithiothreitol, 0.5mM EDTA and 10% glycerol). After em
ometer (Thermo Scientific). 20min, samples were exposed to UV light (254nm) for ic
Wefirstassayedeffectsofeachcompoundonthetranscrip- 10min, digested with 50U of RNase T1, and subjected to .oup
tion activity and on stabilities of the luciferase mRNA and SDS–PAGE (10% gel) and autoradiography. Immunoprecipi- .c
o
proteinusingpcDNA3.1-Lucthatcodes forthenascentfirefly tations were carried out under the same conditions but in m
luciferase and harbors no chimeric exon. We normalized the 40ml. After RNase T1 treatment, we added 360ml of IP /hm
firefly luciferase activities with the cotransfected Renilla buffer (0.1% SDS, 1.1% Triton X-100, 1.2mM EDTA, g/a
luciferase activities. We repeated the experiments five times 16.7mM Tris–HCl pH 8.1, and 167mM NaCl) and 80ml rtic
for each compound. The coefficients of variation (CV%) of protein G-Sepharose (GE Healthcare) precoated with yeast le
-a
five assays were 9.4+4.0% (mean+SD) for all the 960 tRNA. After eliminating the protein G-Sepharose, we incu- b
s
compounds.Wenextperformedduplicateassaystodetermine bated the mixture at 48C for 30min and added 5mg of tra
c
the effect of each compound on splicing of CHRNA1 anti-PTB antibody (ZYMED Laboratory) or isotype-matched t/1
exon P3A harboring IVS3-8G.A (pcDNA3.1-Luc-mtP3A) controlantibodytothesupernatant.Samplesweremixedover- 8/7
by measuring the relative luciferase activities in the presence nightat48Cbeforetheadditionof60mlproteinG-Sepharose. /1
2
of each compound. To estimate the effect of each compound We mixed the sample for additional 30min at 48C. After the 29
on splicing of exon P3A, the relative luciferase activity of beads were washed five times with the IP buffer, bound pro- /67
3
pcDNA3.1-Luc-mtP3A was normalized by dividing it by that teins were eluted and subjected to SDS–PAGE (10% gel) 5
3
ofpcDNA3.1-Luc.ThemeanandSDofthenormalizedrelative and autoradiography. 3 b
luciferase activities of 960 compounds was 1.05+0.20, y g
whereas the mean and SD of 192 negative controls without ue
Surface plasmon resonance analysis s
any compound was 1.00+0.11. Eighty most effective com- t o
pounds with normalized relative luciferase activities of 1.21 We measured the binding affinities of PTB for the wild-type n 2
or higher (mean and SD, 1.36+0.40) were subjected to a and mutant CHRNA1 ISS motifs by surface plasmon reson- 8 M
secondroundofscreeninginduplicate.Thenormalizedrelative ance using Biacore 3000 (GE healthcare). The synthesized a
rc
luciferaseactivitiesofthe80compoundsinthesecondroundof RNA probe were 50-UUUCUCCUUUUCUGUGGRUGGAC h
2
screening ranged from 0.63 to 1.92 with the mean and SD of AGGGUGACAUGGUA-30 with a biotin tag at its 50 end 0
1
1.16+0.25. (Hokkaido System Science). The underlined R was G for the 9
wild-type probe and A for the mutant probe carrying
IVS3-8G.A. The probe was comprised 25 nucleotides in
Analysis of effects of tannic acid on splicing of minigene
intron 3 and 12 nucleotides in exon P3. The probe was
carrying CHRNA1 exon P3A
immobilized onto a streptavidin-coated sensor chip (GE
Inordertoanalyzetheeffectoftannicacidonsplicingofexon healthcare)ataconcentrationof200resonanceunitsbyinject-
P3A,weemployedthepreviouslyreportedpSPL3-wtP3Ahar- ing0.1pmol/mloftheRNAprobeinHBS-EP(0.01MHEPES
boring a 288-bp genomic fragment spanning IVS3-123 to pH 7.4, 0.15M NaCl, 3mM EDTA and 0.005% P20).
IVS3Aþ90 of CHRNA1 (21). We engineered IVS3-8G.A The human PTBP1 cDNA was cloned into a pGEX6P1
to make pSPL3-mtP3A. Transfection into HEK293T cells, vector (GE Healthcare) to make a GST fusion protein in bac-
RNA isolation and RT–PCR were performed as described teria. A fresh colony of Escherichia coli BL21 transformed
(21).WealsoquantifiedthefractionoftheP3A(2)-transcript withpGEX6P1-PTBP1wasgrownin5mlofLBmediumcon-
by estimating the absolute copy numbers of P3A(þ)- and tainingampicillinat378Cfor12h.Theculturewastransferred
Human Molecular Genetics, 2009, Vol. 18, No. 7 1235
to200mloftheLBplusampicillinbroth,andgrowntoOD into pGL3-Basic vector encoding the firefly luciferase gene
600
of 0.1 at 378C. Isopropyl-b-D-thiogalactopyranoside (IPTG) (Promega). To delete three AP2 sites and two Sp1 sites from
was added to a final concentration of 0.5mM, and the the PTBP1 promoter region, we performed three and two
culture was grown for another hour. The bacteria were har- rounds of the QuikChange site-directed mutagenesis kit,
vested, resuspended in 10ml of PBS and lysed by sonication. respectively. We transfected HEK293T cells in a 24-well
Bacterialdebriswasprecipitatedbycentrifugationat10000g. plate with 0.3mg of the pGL3 reporter vector and 0.1mg of
We applied the supernatant onto a glutathione-Sepharose 4B phRL-TK vector (Promega) using FuGENE6. Sixteen hours
column (GE Healthcare), washed the beads with PBS and after transfection, we removed the transfection medium and
eluted the bound protein with 10mM glutathione. added 0–80mM of tannic acid (US Pharmacopeia and Micro-
SourceDiscoverySystems).Weharvestedcells24morehours
and measured the firefly and Renilla luciferase activities. We
Co-immunoprecipitation (Co-IP) to examine the binding
performed all assays three or more times, and measured the
of PTB and hnRNP H D
luciferase activities in duplicate. o
w
We employed the Nuclear Complex Co-IP kit (Active Motif). nlo
Weincubated250mgofanuclearproteinextractofHEK293T ad
e
cells with 3mg of anti-hnRNP F/H antibody (Santa Cruz) or ACKNOWLEDGEMENTS d
isotype-matched control antibody in 500ml of a low salt IP fro
The authors thank Keiko Itano for her technical assistance. m
buffer (Active Motif) overnight at 48C. We added 60ml of h
protein G beads (GE Healthcare) and incubated for an Conflict of Interest statement. None declared. ttp
s
additional hour on a rotary shaker. Beads were then washed ://a
sixtimes with the low salt IPbuffer. The immunoprecipitated ca
d
proteins were dissolved in 20ml of 1(cid:4) SDS sample buffer, FUNDING em
boiled for 5min and analyzed by western blotting using ic
anti-PTB antibody. This work was supported by Grants-in-Aid from the Ministry .ou
of Education, Culture, Sports, Science, and Technology as p.c
well as the Ministry of Health, Labor, and Welfare of Japan, om
Real-time RT–PCR to quantify PTBP1 mRNA and by the National Institutes of Health grant NS6277 as /h
m
ForquantificationoftheexpressionlevelofPTBP1mRNAin well as by a Muscular Dystrophy Association research grant. g/a
HEK293T cells, we employed Hs00259176_m1 for human rtic
PTBP1 (TaqMan Gene Expression Assays, Applied Biosys- le
-a
tems) with the Premix Ex Taq (Takara Bio) in Mx3000P. REFERENCES b
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