Rat lung contains a developmentally regulated
manganese superoxide dismutase
mRNA-binding protein.
H Fazzone, … , A Wangner, L B Clerch
J Clin Invest.
1993;
92(3)
:1278-1281.
https://doi.org/10.1172/JCI116700
.
It has become increasingly clear that RNA-binding proteins play an important role in the
regulation of gene expression. The presence in rat lung of a specific, redox-sensitive
catalase RNA-binding protein was recently reported (Clerch, L. B., and D. Massaro, 1992. J.
Biol. Chem. 267:2853). In order to determine if specific manganese superoxide dismutase
(MnSOD) RNA-binding proteins exist, we tested whether protein in rat lung extract would
bind to 32P-labeled MnSOD RNA. Using a gel mobility shift assay we show rat lung protein
forms specific complexes with a 216 b fragment of the 3' untranslated region of MnSOD
RNA and the binding requires the presence of free sulfhydryl groups. Competition studies
indicate MnSOD RNA-binding protein is different from catalase RNA-binding protein.
Furthermore, unlike catalase RNA-binding protein, rat lung MnSOD RNA-binding protein
activity is developmentally regulated; there is less MnSOD RNA-protein binding activity in
adult rat lung extract compared to prenatal or neonatal rat lung extracts. We conclude the
lung contains developmentally regulated MnSOD mRNA-binding protein that is redox
sensitive.
Research Article
Find the latest version:
Rat Lung Contains
a
Developmentally
Regulated
Manganese Superoxide Dismutase mRNA-binding Protein
HilaryFazzone, Angelique Wangner, and LindaB.Clerch
LungBiology Laboratory, Department ofPediatrics, Georgetown University Medical Center, Washington,DC 20007
Abstract
It has become
increasingly
clear that
RNA-binding proteins
play an important role in the regulation of gene expression. The
presence in
ratlung of
aspecific, redox-sensitive catalase
RNA-binding
protein
wasrecently
reported (Clerch,
L.
B., and
D. Massaro, 1992. J. Biol. Chem.
267:2853).
Inorder
todeter-mine if
specific
manganese
superoxide
dismutase
(MnSOD)
RNA-binding
proteins exist,
wetested whether
protein
in
ratlung extract would bind
to32P-labeled MnSOD RNA.
Using
agel
mobility shift
assayweshow
ratlung protein
forms
specific
complexes with
a216 b
fragment
of the 3' untranslated
region
of MnSOD RNA and the
binding requires
the presence of free
sulfhydryl
groups.
Competition
studies indicate MnSOD
RNA-binding
protein
is different from catalase
RNA-binding
protein. Furthermore, unlike catalase
RNA-binding
protein,
ratlung MnSOD
RNA-binding
protein activity
is
developmen-tally
regulated;
there is less MnSOD
RNA-protein binding
ac-tivity in adult
ratlung
extractcompared
toprenatal
orneonatal
rat
lung
extracts.We conclude the
lung
contains
developmen-tally
regulated
MnSOD
mRNA-binding
protein
that is redox
sensitive.
(J. Clin.
Invest. 1993.
92:1278-1281.) Key
words:
antioxidant
enzymes *
posttranscriptional
regulation
* gene
ex-pression * redox
Introduction
The
antioxidant
enzymes
including catalase, MnSOD,
CuZn-SOD,
and
glutathione
peroxidase (GP),' perform
aninvalu-able function
by reducing
the cellular concentrations ofsuper-oxide
and
hydrogen peroxide ( 1).
These toxic moieties areformed
during
normal oxidative metabolism
but theirforma-tion increases
during periods
of oxidantstress(2-4).
Anim-portant aspect of antioxidant enzymes
is thatthey
aremutually
supportive;
catalase and GP
protect the SODsagainst
inactiva-tion
by
hydrogen
peroxide
and, reciprocally
the SODs protect
catalase and GP
against
inactivation
by superoxide (5).
Al-though
the antioxidant enzymes work
together
inacoordinate
way, the
mechanisms
responsible
for the
regulation
of
their
Addresscorrespondenceto Dr. LindaB.Clerch, Preclinical Science
Building (GM-12),GeorgetownUniversityMedical Center, 3900
Res-ervoirRoad,NW,Washington,DC20007.
Receivedfor publication 19October1992and inrevised form23
March 1993.
1.Abbreviations usedinthispaper: GP,glutathione peroxidase;2-ME, 2-mercaptoethanol; NEM,N-ethylmaleimide;UTR, untranslated
re-gion.
gene expression
candiffer. For example, the concentration of
lung MnSOD mRNA, but not that of catalase or GP, increases
during exposure of adult
rats to 48 hof
>95% 02(6).
Further-more, the level of regulation may be developmentally
depen-dent;
ratlung catalase
mRNAconcentration increases during
late
gestation
andduring exposure of
theneonate
tohyperoxia;
however, catalase
mRNAconcentration is regulated at the
level of transcription during late gestation but by increased
mRNA
stability in neonatal rat lung during hyperoxia (7).
Since
RNA-protein binding
canalter
mRNAstability,
onepos-sible mechanism responpos-sible for altered catalase mRNA
stabil-ity during exposure of neonatal rats to hyperoxia would be the
interaction between catalase mRNA
and acatalase
mRNA-binding
protein.
Rat lung has been shown tocontain
aredox-sensitive
protein that specifically interacts with catalase RNA
to
form RNA-protein complexes (8). In addition, this
protein-binding activity inversely correlates
withcatalase mRNA
stabil-ity, suggesting that oxidation-induced disassociation ofcatalase
mRNA
from its binding protein might result in increased
stabil-ity of the mRNA (7, 8). That is, the catalase RNA-binding
protein could function
as atrans-acting negative
regulator of RNAstability.
Using
agel
mobility
shift assay,
we nowshow rat lung
con-tains MnSOD RNA-binding protein and
that thebinding
be-tweenthe
RNAandprotein requires the presence of free
sulf-hydryl groups. Competition studies indicate MnSOD
RNA-binding
protein is different from catalase RNA-binding
protein. Furthermore, unlike catalase RNA-binding protein,
rat lung MnSOD RNA protein-binding activity is
developmen-tally
regulated;
ourdata indicate
that the activity is greater in
fetal and neonatal
ratlung, compared
toadult
ratlung.
Methods
Animals.Weusedspecific pathogen-freeSprague Dawley rats obtained fromZivic-MillerLabs, Inc. Allison Park, PA, or from Harlan Farms,
Indianapolis, IN.Theyweremaintained in our Animal Care Facilities
ona12-hlight-darkcycle. Neonatal rats were designated to beI-d old theday afterbirth;theywereraised 10 perlitter, and the litters were
adjustedtothatsize within 12 h of birth. For the hyperoxia study, rats wereexposed in
3.4-ft3
plastic chambers in which02
(> 95%),CO2(<0.1%), temperature (22-25°C), and humidity (40-60%) were
monitored.Air-breathingratswereexposed in identical chambers in which airflowedfrom a compressed air generator. To prevent
02-in-duced lung damagein the nursingdams, they werealternateddaily
betweenlitters of pups exposedtoair and
02.
Adultrats wereallowed food andwateradlib. and pupswereallowed freeaccesstothe dams.Rats werekilled between 9a.m.and 11 a.m.byintraperitoneal injec-tionofpentobarbitalsodium ( - 200 mg/kg), followed by exsanguina-tion.
Preparationofcell extracts. The lungs of fetal, neonatal, or adult ratswereperfusedfree of blood, excised, and homogenized ina25mM
Tris
buffer,pH7, containing 0. 1mMEDTA,1%Tritonx-100, 40mMKCl,
10 ug/mlleupeptin, 0.1mMphenylmethylsulfonylfluoride,and 0.2U/Ml
aprotinin.Thelunghomogenatewascentrifugedat12,000 gJ.Clin.Invest.
© The AmericanSocietyforClinical Investigation, Inc.
0021-9738/93/09/1278/04 $2.00
for 15min at4VC. The supernatant fluid (hereafterreferredtoas lung extract) wasstored at-70'C.Theproteinconcentration of the lung extract was measured using the Coomassie Plus assay from Pierce Chemical Co., Rockford, IL, with BSA as a standard.
Preparation oflabeled and unlabeled cRNA probes. The RNA probe usedfor MnSOD RNA binding experiments was derived from a Stul-EcoRIrestriction fragment ofa ratliver MnSOD cDNA-Ikindly provided by Dr. Ye-Shih Ho, Wayne State University, Detroit, MI (9). The 885b Stul-EcoRI restriction fragment, which encompasses the coding region for47carboxyl-terminal amino acids and 744 basesof
the3' untranslatedregion,wassubclonedintoapGEM-7Zvector (Pro-megaBiotec,Madison,WI). The DNA sequenceofboth strandsofthis recombinantplasmid (designated RMS-12) was determinedusingthe
Sangerdideoxy-nucleotidechaintermination method. In orderto de-limit theregionsof MnSOD RNA that are involved inbindingprotein,
theplasmid was made linearusingtherestriction enzymes,XbaI, PstI,
orNdeI, and labeledsensestrandtranscriptswerepreparedusingSP6 RNApolymerase and[32P]CTP accordingtoPromegaBiotec's proce-dure for invitrosynthesisofhigh specific activity radiolabeledRNA probes. The resultantMnSOD cRNAsaredesignated RMS-E, RMS-P,
andRMS-N,respectively.Followingelectrophoresisina4%
nondena-turingpolyacrylamide gel,the
transcripts
wereexcised from thegel
and elutedovernightin 0.5 Mammoniumacetateand 1 mM EDTA. The probe wasprecipitatedby ethanol andresuspendedin 20 MlH20).Unlabeled MnSOD cRNA forcompetition experimentswas pre-pared from thesamerecombinantplasmidusedtoprepare the labeled MnSOD cRNA probe (RMS-E)accordingtoPromega Biotec's proce-dureforthesynthesisof largequantitiesof RNAtranscripts.The unla-beledcompetitors,catalase cRNA and lectin-14K(ratlung
f3-galacto-sidelectin,subunitM,
14,000)cRNAwerepreparedasdescribedprevi-ously from plasmids containing the catalase or lectin-14K cDNA inserts (8). Theconcentrationsofunlabeled cRNAspecieswere
mea-suredspectrophotometricallyby absorbanceat260nmand usedat a
-100-foldmolar excess comparedtothe
32P-labeled
MnSOD cRNA probe.RNA-proteinbinding assay. Thebinding assaywasperformedby
incubatinglung extractcontaining20-30,gprotein with - 2X 104
dpm of
32P-labeled
MnSOD cRNA probe in 30Ml ofbinding buffer containing5%glycerol, 40mMKCl,
3 mMMgCl2, 10mMHepes, pH 7.6.Thesolutionswereincubatedat25°C for 30 min.Tl
RNase( 1U)(Gibco BRL, Gaithersburg, MD), which degrades single-stranded
RNAthatis unprotected because it is not bound to protein, was then added andincubation continuedfor10 min.Heparinsulfate(6
Mg/Ml),
which removesnonspecificallyboundproteins from RNA, was added andincubation continued for 10 min. A gelloading buffer of50% glycerol and 1%bromophenol bluewasadded to thereaction mixtures
thatthen underwentelectrophoresisat 14V/cm inalowcrosslinked
4%polyacrylamide gel (acrylamide: bis-acrylamide 60:1) containing
89 mMTris-borate,pH8.3,and 2 mM EDTA.Afterelectrophoresis,
the gelwastransferredtofilter paper,dried,and exposedtox-ray film
(Hyperfilm;AmershamCorp.,Arlington Heights,IL)at-70°Cwith
anintensifyingscreen.In someexperiments densitometry usinga
scan-ning densitometer (Image Quant version 3.2; Molecular Dynamics, Inc.,Sunnyvale, CA)wasused toquantitatebinding activity.
Proteinase, competition, and oxidation-reduction experiments.
Proteinase experiments were done by pretreating lung extract with 500
Mg/ml proteinaseK for 1 hat37°C.In competition experiments,
bind-ingreactionmixtureswereincubatedwith an unlabeledRNAspecies (MnSOD cRNA, catalase cRNA, lectin-14K cRNA, or yeast tRNA) for 30 minat25°C before theaddition of labeled MnSOD probe.
Re-dox-sensitivity experimentswereperformedby incubation of reaction mixtures with 100 mMdiamide (anoxidizingagent);
1O
mM N-ethyl-maleimide (NEM),analkylating agent;or2%2-mercaptoethanol (2-ME), areducing agent; for 15 min at 25°Cbefore the addition of labeledMnSOD RNA probe.Statisticalanalysis.Fordensitometrydatagiveninarbitraryunits,
the values for individual animals were averaged per experimental
group andthe SEMof thegroupwascalculated. The
significance
of thedifference between two groups was obtained using an unpaired t test
analysis (10).
Results
Using
the
gel mobility
shift assay,
wefoundasingle major
band(arrow) in the
binding
reaction between
ratlung
extractandthe 885b 32P-labeled MnSOD
cRNAprobe designated
RMS-E(Fig. 1, lane 1).
Additional
bands
(arrowhead) representing
complexes
of greater mobility
wereoccasionally
observed;
these
bands appear less intense than the single, consistently
observed band
indicated by the arrow. The faster migrating
bands may represent degradation products of
thelarger
com-plex. When lung extract was pretreated with proteinase
K,the
RNA-protein complex was not found, indicating
thematerial
bound
to theMnSOD
RNA is aprotein
(Fig.
1A,
lane2).
Treatment
of lung
extractwith NEM,
anagent that
alkylates
and
therefore blocks free sulfhydryl groups, or with diamide,
an
agent
that oxidizes free sulfhydryl groups
to formdisulfides,
eliminated MnSOD RNA-protein complex formation (Fig.
1B,
lanes 2 and 3, respectively
vs. lane1,
a controlreaction
inthe absence of alkylation or oxidation). The
additionof 2-ME,
a
thiol reducing agent, enhanced band intensity (Fig.
1B,
lane 4 vs. lane 1). These resultsdemonstrate
theredox-sensitivity of
the
MnSOD mRNA-protein interaction. The presence
of 2-MEin
thereaction mixtures
causedrestoration ofbinding
activ-ity following
itselimination
with diamide
(data not shown).This sulfhydryl switch mechanism,
bywhich RNA-protein
binding is abolished by oxidizing agents
but is restoredby
re-ducing agents,
is similar to thatpreviously observed for
thecatalase mRNA-protein interaction (8). These results
are ofparticular interest because they indicate the
oxidation-reduc-tion state of proteins
thatbind
toantioxidant
enzymemRNAs
A
"Is
wo 8
0 on 8
us
-proteinaseK
N
+
1
-1 2 1 2 3 4
Figure 1. Complexformation between MnSOD RNA and lung pro-tein issensitive to redox agents. (A) One major complex (arrow) is formed during the binding reaction of 32P-labeled MnSOD RNA probe(RMS-E) with lung protein extract (lane 1). The arrowhead points to a lessintense complexof faster mobility.Lane2 shows the lackof complex formation when the lung extract was pretreated with proteinase K (500Mg/ml). (B) One major complex (arrow) is formed when neither reducingnoroxidizing agentwasaddedto the reaction mixture (lane 1). Preincubation of the lungextractwith either10 mM NEM (lane 2) or 100mMdiamide (lane 3)completely
may have an important role
regulating
antioxidant enzyme geneexpression during the lung's response to oxidant stress.To determine the specificity of the RNA-protein interac-tion weperformed competition studies in which lung extract was preincubated with unlabeled RNA before the addition of radiolabeled MnSOD RNA. In the absence of competitor, the radioautograph shows one major complex (arrow) was formed after incubation of radiolabeled MnSOD RNA with lung pro-tein extract(Fig. 2, lane 1). The MnSOD RNA-protein com-plex demonstrated in lane I wasspecifically inhibited by petition with unlabeled MnSOD RNA (Fig. 2, lane 2) but
com-plex formation was not eliminated by preincubation with
tRNA, catalasecRNA, or lectin-14K cRNA (Fig. 2, lanes 3, 4, and 5, respectively). In cross-competition experiments using radiolabeled catalase RNA and unlabeled excess MnSOD RNA, we
found
MnSOD did not inhibit formation of catalase RNA-protein complexes (data not shown). The results of thecompetition
studies indicate that binding observed withMnSOD
RNAis specific
and the MnSOD RNA-bindingpro-tein is different from
thecatalaseRNA-binding
protein. We usedunique restriction
endonuclease sites (PstI and NdeI)within
the StuI-EcoRIfragment
of MnSOD cDNA- 1 to delimit the region of MnSOD RNA involved in proteinbind-ing.
Thesize
andorientation of
the MnSOD cRNA probes aredepicted
inthe
diagram
inFig.
3 A.Of
thethree32P-labeled
cRNA
probes
generated by invitro transcription,
we foundRMS-E
andRMS-P,
but notRMS-N,
contain cis elements that bindlung protein (Fig.
3B).
Theprotein binding activity to both RMS-E and RMS-P wasenhanced in vitro by the addition of 2-ME (Fig. 3 B). These findings indicate protein binding occurs in a216-basefragment
of the 3' untranslatedregion
(3'PS~~~i
0competitor 0 "4
1 2 3 4 5
Figure2. MnSOD
RNA-binding protein
isdifferent from catalaseRNA-binding
protein.Onemajor
MnSODRNA-protein binding
complex
(arrow)
is formed whenratlung
extractis incubated with MnSOD cRNA(RMS-E)
in the absence ofcompetitor
RNA(lane 1).MnSODRNA-protein
binding
isinhibitedby
incubation withexcessunlabeled MnSOD cRNA(lane
2).
Thereisnoinhibition ofbinding
in thepresenceofnonspecific
yeasttRNA(lane3)
orin thepresenceofexcessunlabeled catalase cRNAorlunglectin-14K cRNA (lanes4and 5,respectively).
A
RMS-E SI _ _ _ _ _ _
(88Sb) 577
S
RMS-N 5'V 3'
S N
RMS-P . 3'
S p
RMS-NP
(216b) B
PMOE
3,
E
5' 3'
r
--N P
INS-E
2- +
RNS-P
+
INS-N
1 2 3 4 5 6
Figure 3. The MnSODRNA-protein binding element is located ina
216-basefragment of the 3'UTR.(A)Diagram of MnSOD cRNA probes generated by in vitro transcription after linearization of plas-mid RMS- 12 by restriction endonucleases. Thedesignatednameand length of the probe is given on the left. The 5' to 3' orientation is shown and theMnSODcoding regionisdepicted bythe cross-hatched bar. RMS-NP(dashed line)represents the deducedprotein binding
ciselementof the MnSOD 3' UTR. S, StuI; E, EcoRI; N, Ndel;P, PstI.(B) Gel mobilityshift assay using RMS-E (lanesIand2), RMS-P(lanes 3 and 4), and RMS-N (lanes 5 and 6). Thebinding
reactionwas donein the absenceof 2-ME (lanes 1, 3, and 5) and in the presence of 2% 2-ME (lanes2, 4, and 6). Themajor complex
formed between 30 ,ug of neonatallungproteinand RMS-E and RMS-P, but not RMS-N, is indicated by thearrow.
UTR)
between the NdeI and PstI restriction endonucleasesites. This
region, which
wedesignate
RMS-NP, is located
111 bases downstream of the translation stop codon(Fig.
3A).
RMS-NP
is contained within
the339-base NdeI-EcoRV
frag-ment
of
3' UTRshown
by
Hurt et al. to bepresent infour
ratMnSOD RNA
species (3.8, 2.7, 2.2,
and1.3
kb)
thatoriginate
asa
result of
alternatepolyadenylation ( 1).
MnSOD
mRNAprotein
binding
appears to bedevelopmen-tally
regulated.
Wefound similar band
intensity
on thera-dioautograph of
thebinding reaction between
MnSODRNAand
lung
extractsfrom fetal
andneonatal
rats(Fig.
4A,
lanes I to4). Complex
formation with adultlung
extract(Fig.
4A,
lane5) appears as a band
of much
lessintensity
than
thebands
formed
withperinatal
extracts(Fig.
4A,
lane 5vs.lanes 1to4).
We
found
nodifference in
RNA-protein
binding activity
be-tweenlung extractfrom
7-d-old ratsexposed
to 72 h>95%02
and
air-breathing
7-d-old (
1.97±0.20SE,
n=4,
and 1.89±0.22SE,
n=3,
respectively). Fig.
4 Bshows
thecomplexes
formed
using
lung
andbrain
extractsfrom
three7-d-old
neonatalratsand three
adult rats.Using scanning densitometry,
wefound
significantly
greaterMnSOD
RNA-binding
activity
inneona-tal
lung
extractthan in adultlung
extract(2.58±0.26
SE vs.0.73±0.01
SE,
P<0.005).
Wedid
notfind
asignificant
differ-ence in
MnSOD
RNA-binding activity
between neonatalA 0 N
-fNre
lung
~~~~~~~~~~~~~~:.
0
0e
0 0 3 4 5
JB4
lung
bindingactivity in adult extracts, perhaps duetooxidation of the binding protein. Other possible explanations for less MnSOD RNA-binding activity in adult lungs include: (a) de-creased concentration of the binding protein in adult lung comparedtoperinatal lungs and (b) decreased RNA-binding affinity of the protein in adultextractscomparedto neonatal
extracts.We havenotdistinguished between these possibilities
atthistime.
Inorder to
determine
the in vivo function of the MnSODRNA-binding
protein, further studies arerequired.
However, we speculate that MnSOD RNA-binding protein may have a role during development as a negative regulator of MnSOD mRNAstability.
Webasethis
speculation
onprevious
pub-lished
workshowing
thatlung
MnSOD RNA in adult rats hasalonger half-life (8.2 h±0.8 S.E.) than MnSODRNA in
neona-tal
lung
(5.5 h±O.ISE,
P <0.025)
(6, 12). The increased stability of adult rat lung MnSODmRNAcorrelates withde-creased RNA-binding protein activity, suggesting the binding
protein
may be a trans-acting negative regulator of stability. Future work will be focused on isolating the MnSOD RNA-binding protein to directly test its function.Acknowledaments
brain
day 7 adult day 7 adult
IF-
:1
1z $1 2 3 4 5 6
...._...
.
_-__ 9 1 I 2
Figure 4. Rat lung MnSODRNA-protein binding activity is
develop-mentally regulated. (A) The MnSOD RNA binding reaction was per-formed using the RMS-P probe with30-,uglungprotein extract from
rats atgestation day 20, gestation day 22, postnatal day 1, postnatal day 7, and adulthood (250-300 g) (lanes I to 5, respectively). The
arrowpointstothemajor RNA-proteincomplex. Theposition of free unbound probe is indicated. (B) MnSOD RNA-protein binding
activ-ityis developmentally regulated inratlung but not in rat brain. The MnSOD RNAbinding reactionwasperformedusing the RMS-P probe and30
,g
ofproteinextractfrom7-d-oldratlungs (lanes 1-3), adultratlungs (lanes 4-6), 7-d-old ratbrain (lanes 7-9), and adult rat brain (lanes10-12).
5.60±1.97
SE, respectively).
These dataindicate
rat lung MnSODRNA-protein
bindingactivity
is developmentally reg-ulated andthis
developmental regulation is, at least in part,tissue specific.
The cause andsignificance of
the decreasedbind-ing
activity observed
in adultlungs compared
toperinatal
lungs
is
unknown. Itis
possible
that thereis
aninactivation
ofWethank Dr. Donald Massaro,for helpful advice during this work and for criticalreadingof thismanuscript.
This research was supported by grants from the American Lung Association and the National Institutes of Health (20366 and HL-47413). H. Fazzone is the recipient ofaGeorgetownUniversity Medi-cal StudentSummer Research Award. L. B. Clerch isaParkerB.
Fran-cis Fellow in Pulmonary Research.
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It's"
1.