a high yield affinity purification method for specific rna- binding

Nucleic Acids Research, Vol. 18, No. 1 1990 Oxford University Press

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor fromhuman placenta

Barbara Neupert, Nancy A.Thompson, Christine Meyer and Lukas C.Kuhn*Swiss Institute for Experimental Cancer Research, Genetics Unit, CH-1066 Epalinges, Switzerland

Received October 26, 1989; Revised and Accepted November 28, 1989

ABSTRACT

We describe a simple method for the affinitypurification of specific RNA-binding proteins. DNAsequences corresponding to the protein-binding siteof the RNA are subcloned into an in vitro transcriptionvector between the T7 viral promoter and a poly(A)track. A polyadenylated RNA transcript is bound topoly(U)-Sepharose and subsequently incubated with acellular extract prepurified on heparin-agarose.Specifically adsorbed proteins are recovered in highyield and purity from the affinity matrix by high saltelution. Using this method we isolated the ironregulatory factor (IRF), a cytoplasmic protein whichbinds to specific palindromic elements in the 5' and 3'untranslated sequences of ferritin and transferrinreceptor mRNA, respectively. Activation and bindingof this regulatory factor correlates with increasedtransferrin receptor mRNA stability and inhibition offerritin translation. The purified factor from humanplacenta migrates as a monomer in gel chroma-tography, but is present in equimolar amounts of twoproteins with molecular weights of 95 and 100 kDawhen analysed by SDS/PAGE. The two proteins arehighly related as judged by the identity of theirisoelectric points and their specificity to form RNA-protein complexes.

INTRODUCTION

The iron regulatory factor (IRF), also named iron-responsiveelement binding protein (IRE-BP) or ferritin repressor protein(FRP), is a cytoplasmic mRNA-binding protein whose functionis of central importance in cellular iron homeostasis. Two distinctpost-transcriptional regulatory mechanisms that adjust theexpression of the main effector proteins in iron metabolism,ferritin and transferrin receptor (TR) (1—8), are coordinatelycontrolled by the activity of IRF (9-11). As first recognizedby Leibold and Munro (9), IRF binds specifically to a palindromicsequence element in the 5' untranslated region (UTR) of mRNAsencoding the iron storage protein, ferritin (9,12). This interactioninhibits translation both in vivo (2-5) and in vitro (13 — 15).Similar IRF-binding sites, also termed iron-responsive elements

(IRE), have subsequently been located in the 3' UTR of TRmRNA (10,11,16,17), where they map to sequences that arenecessary for iron-dependent regulation of TR mRNA stability(6,7). IRF-binding to these IREs correlates with the inhibitionof TR mRNA decay. This post-transcriptional regulatorymechanism requires the presence of a second structural element,a stem-loop structure, which is thought to confer instability tothe mRNA in the absence of IRF-binding (10). Thus, besidescontrolling iron storage through inhibition of ferritin synthesis,IRF also increases TR expression and iron uptake via receptormediated endocytosis of transferrin. IRF is therefore consideredto act as a feedback regulator of intracellular iron levels (10,18).This contention is supported by the finding that the RNA-bindingactivity of IRF itself is modulated as a function of cellular ironlevels and is highest when cells are deprived of iron (9,10,12).It remains unknown whether heme (19), iron itself (20) or aniron-containing enzyme are required for IRF activation. In vitroexperiments did not reveal a direct involvement of iron as acofactor of IRF binding to RNA (10,21). Recent evidence rathersuggests that IRF activation is due to reversible oxido-reductionof a sulfhydryl group in the protein (21). Isolation of IRF isclearly required to obtain further information about its activationand physical interaction with the characteristic IREs in mRNAs.

In the present study we have purified IRF from human placentato homogeneity. To this end we have developed a simple newaffinity purification procedure which has the potential of beinggenerally applicable for isolating RNA-binding proteins. In vitrotranscribed polyadenylated RNA is bound to a poly(U)-Sepharoseand incubated with cellular extracts. The specifically adsorbedproteins are recovered by elution with 1 M KC1. This purificationscheme yields IRF with a substantially higher recovery than arecently published affinity method (22) and is considerably fasterthan classical isolation procedures (15).

MATERIALS AND METHODS

Plasmid Constructions

Plasmid pSPT-TR21 has previously been described (10) and isderived from a 3' deletion mutant of the hTR cDNA clone pcD-TR1 (7,23) subcloned into the pSPT18 vector (Boehringer,Mannheim, FRG) behind the T7 promoter. Transcription of this

* To whom correspondence should be addressed

51

Downloaded from https://academic.oup.com/nar/article-abstract/18/1/51/1036848by gueston 19 March 2018

52 Nucleic Acids Research

plasmid in vitro by T7 RNA polymerase generates an RNA whichincludes all 5 iron-responsive elements (IREs) from the hTR 3'UTR. pSPT-fer contains an oligonucleotide that specifies the 5'IRE of human ferritin heavy chain mRNA, bases 31 -58 (10,24).pSPT-TR35 contains hTR 3' UTR sequences between position4048 (25) and the BamHI site 29 bases 3' to the poly(A) regionin the pcD-vector (26). Transcripts from this plasmid arepolyadenylated and lack 3' regulatory elements. To obtain apolyadenylated transcript equivalent to pSPT-TR21, weconstructed a new vector, pSPT-PA, which carries theXbal/BamHI fragment from the 3' end of the hTR cDNA clonepcD-TRl (23) subcloned between the corresponding sites inpSPT19 (Boehringer, Mannheim, FRG). This fragment is about270 bases in size and contains the following sequence:5'TCTAGAACTTGCATGACCTTTACTGTGTTAGCTCTT-TGAATGTTCTTGAAATTTTAGACTTTCTTTGTAAACA-AATGATATGTCTTATCATTGTATAAAAGCTGTTATGT-GCAACAGTGTGGAGATTCCTTGTCTGATTTAATAAA-ATACTTAAACACTG (A)84 GTACCTTCTGAGGCGG-AAAG AACC AGCGG ATCC.

The insert from pSPT-TR21 (bases 3421-3597,3848-4048of pcD-TRl, numbered according to Schneider et al. (25) wassubcloned between the T7 promoter and the Xbal site of pSPT-PA. The resulting plasmid is referred to as pSPT-TR38.

Preparation of in vitro transcriptsAll RNA transcripts were generated by in vitro transcription (27)with T7 polymerase using linearized plasmid DNA as a template.RNA transcripts of high specific activity were synthesized from1 ng DNA in the presence of 100 /*Ci [a-32P]CTP (800Ci/mM)(Amersham, Buckinghamshire, UK), 0.5 mM ATP, GTPand UTP (Pharmacia, Uppsala, Sweden) and 20 units T7 RNApolymerase (Boehringer, Mannheim, FRG) in a 20 /tl reactionvolume. Samples were incubated for 1 hour at 40°C. The specificactivity of transcripts under these conditions is 1.3 X 109 dpm/^gRNA. For large scale synthesis of unlabelled RNA, transcriptionreactions were performed with 10 ng plasmid DNA in thepresence of 1.5 mM ATP, CTP, GTP, and UTP (Pharmacia,Uppsala, Sweden) and 60 units T7 polymerase (Boehringer,Mannheim, FRG) in a final volume of 100 /i\. Samples wereincubated for 2 hours at 40°C. To increase the yield, the reactionwas continued for another 2 hours after addition of a fresh mixtureof 50 /tl 1.5 mM rNTPs together with 40 units T7 polymerase.RNA transcripts with low specific activity were generated by thesame procedure, but included 120 /*Ci [a-32P]CTP (800Ci/mM) in the reaction. Specific activity of transcripts is1.3 X106 dpm/jtg. Unincorporated nucleotides were removed ona Sephadex G-50 column.

Gel-Retardation AssaysAnalysis of RNA-protein interactions were performed asdescribed by Leibold and Munro (9) incubating a molar excessof 32P-labelled RNA transcript with aliquots from proteinfractions in a 20 yX reaction volume. Binding was carried outfor 10 min at room temperature. Heparin was added to a finalconcentration of 5 mg/ml for another 10 min. No RNase Tldigestion was required using the RNA probe from pSPT-fer.Binding assays with purified IRF were performed in the absenceof both RNase Tl and heparin. RNA-protein complexes wereresolved in 4% non-denaturing gels as described (28). Whereindicated, IRF was either reduced with /3-mercaptoethanol oroxidized with azodicarboxylic acid bis[dimethylamide] (Sigma,St.Louis, MO), a thiol-oxidizing agent, known as diamide (29).

Preparation of human placental extractsFresh human placenta was dissected free of connective tissue,cut into small pieces, extensively washed with ice cold PBS andground in a manual tissue grinder. All steps were performed at4°C. The tissue was suspended in an equal volume of a 0.1 M2-morpholinoethanesulfonic acid buffer pH 6.5 supplementedwith 1 mM EGTA, 0.5 mM MgCl2, 3 mM NaN3 and 1 mMPMSF. It was homogenized first in a Kinematica Polytron blender(Kriens, Switzerland) at speed 8 for 12 sec and then passed twicein a motor-driven Potter Elvehjem Tissue Grinder with a teflonpestle. About 500 /tl cytoplasmic extract were recovered bysaving the supernatant of sequential centrifugations at 3000 rpmfor 10 min and 8500 rpm for 15 min in a Sorvall GS-3 rotor,and at 25,500 rpm for 60 min in a TST 28.38 rotor (Kontron,Zurich, Switzerland). The protein concentration was measuredby the protein assay of Bio-Rad (Richmond, CA).

Prepurification of IRFAliquots of placental extract (45 ml) were fractionated on aSephacryl S-300 column (dimensions 19.6 cm2x90cm)(Pharmacia, Uppsala, Sweden), equilibrated with buffer A (10mM Hepes buffer pH 7.5, 40 mM KC1, 3 mM MgCl2, 5%glycerol). Fractions containing IRF activity were pooled andreduced with 2% /3-mercaptoethanol (21) prior to adsorption onHeparin-Sepharose beads (Pharmacia, Uppsala, Sweden)equilibrated in buffer A with 2% /3-mercaptoethanol. The bindingwas done at a ratio of 100 mg protein/1 ml beads under constantshaking for 3 hours at room temperature. The Heparin-Sepharosewas then washed three times with 10 volumes of buffer A andpacked into a column. Bound proteins were eluted with 250 mMKC1 in 10 mM Hepes buffer pH 7.5, 3mM MgCl2, 5%glycerol.

Affinity purification of IRFPoly(U)-Sepharose (Pharmacia, Uppsala, Sweden) wasequilibrated in RNA binding buffer (25 mM Hepes buffer pH7.5, 100 mM KC1), and 0.5 ml beads were packed into a 10ml column. Polyadenylated in vitro transcripts of pSPT-TR35 orpSPT-TR38 (600 or 300 /*g, respectively, in 500 /JL\ RNA bindingbuffer) were applied onto the column at room temperature andrecycled three times. By including 106 cpm of low specificactivity transcript we found that more than 90% of the RNA wasbound under these conditions. The affinity column wasequilibrated in buffer A. In order to permit binding to RNA, theenriched IRF fraction from the heparin column was diluted three-fold in buffer A without KC1. In the course of experiments wefound it necessary to add 30 units RNasin (Promega, Madison,WI) per ml protein solution to preserve the column during theaffinity purification. In addition, to prevent unspecific RNA-protein interactions 5 mg/ml heparin (Serva, Heidelberg, FRG)and 35 jig/ml rRNA (Boehringer, Mannheim, FRG) were addedas competitors. The sample was recycled three times on theaffinity column at room temperature. Under these conditions morethan 95% of the IRF activity was retained. The column waswashed first with 20 volumes of buffer A containing 5 mg/mlheparin and then 40 volumes of buffer A alone. IRF was elutedwith 1 M KC1 in 25 mM Hepes buffer pH 7.5 and an aliquotwas tested for its activity after a 10-fold dilution in buffer Awithout KG. Purified IRF was concentrated and equilibrated withbuffer A on a centricon-30 microconcentrator (Amicon, Danvers,MA) and analysed by SDS-PAGE.


Nucleic Acids Research 53

Analysis of purified IRFAffinity-purified IRF was analysed by fast pressure liquidchromatography (FPLC) on a Superose 12 column equilibratedin 50 mM NH4HCO3 pH 8.0. Protein fractions were analysedby the gel-retardation assay, as well as on silver stained SDS-polyacrylamide gels (Bio-Rad, Richmond, CA). Two dimensionalgel electrophoresis of affinity-purified IRF (5 jtg) was performedaccording to the method of O'Farrell (30).

RESULTS AND DISCUSSION

In previous in vitro experiments we established that at least 4out of the 5 palindromic IREs in the 3' UTR of TR mRNA

PrepurifiedIRF

RNA 5'

Specificallybound IRF

Elution of IRFwith 1 M KCI

Poly (U) -Sepharose

AAAAAAAUUUUUUUUUUU

Figure 1. Schematic representation of the affinity purification of IRF. In vitrotranscribed polyadenylated RNA is bound to poly(U)-Sepharose. Prepurified IRFis specifically adsorbed to the IRE and subsequently eluted with 1 M KCI.

110FRACTION NUMBER

Figure 2. Fractionation of human placenta] extract on a Sephacryl S-300 column.The upper panel depicts the elution profile of protein monitored at 280 nm. Themajor protein peak corresponds to serum albumin. In the lower panel every thirdfraction was analysed by the gel-retardation assay using 10 ii\ protein aliquotsand 106 cpm of the high activity RNA transcript from pSPT-fer.

interact simultaneously and specifically with monomers of IRF(10). We also noticed that 32P-labelled RNA-protein complexeswere highly stable in vitro and did not dissociate upon dilutionof the reaction mixture in the presence of an excess unlabelledIRE-containing RNA (unpublished observation). We thereforedecided to isolate IRF by affinity purification thus takingadvantage of its RNA-binding properties (Fig.l).

Prepurification of IRFThe choice of human placenta as a source of IRF was influencedby our previous finding that this tissue has substantial quantitiesof IRF whose molecular weight and RNA-binding specificity areidentical to the protein present in murine cells (10). Calculationsbased on a 1:1 ratio of the IRE-IRF interaction let us estimatethat the amount of IRF present in placental extracts isapproximately 1 mg per 10 g of protein. In order to preventdegradation of the RNA affinity column by the possible presenceof ribonucleases and also to concentrate IRF in a small volumeprior to its adsorbtion to the affinity matrix, we decided toprepurify IRF on a Sephacryl S-300 column followed byfractionation on Heparin-Sepharose. As expected from ourprevious results using FPLC (10), IRF activity eluted as a singlepeak slightly in front of serum albumin (Fig.2). The main IRFcontaining fractions, 95 to 112, were pooled and adsorbed toHeparin-Sepharose using a batch procedure. Only about 2% ofthe total protein but more than 90% of IRF bound to this matrixand almost none of the IRF dissociated upon repeated washingof the heparin-beads. In an attempt to recover IRF at a specificsalt concentration we found a broad peak of elution between 50and 200 mM KCI (data not shown). In subsequent experimentsIRF was therefore eluted in a single step at 250 mM KCI witha recovery of approximately 70% compared to the original S-300fraction (Table 1). Prior to the Heparin-Sepharose purificationstep we reduced IRF with 2% j3-mercaptoethanol which hasrecently been found to result in the full activation of the RNA-binding activity of IRF (21). We noticed, however, that IRF bindsequally well to heparin-beads in the absence of the reducing agent.

RNA affinity columnThe final step in the isolation of IRF was achieved on a specificRNA affinity column. Methods for affinity purification of RNA-binding proteins have previously been applied to isolate factorsinvolved in RNA splicing (31,32). These methods consist of thein vitro transcription of RNA containing allylamine-uridine whichis subsequently biotinylated and bound to biotin-agarose viaavidin. In our present approach we devised a procedure which

Table 1. Purification of IRF.

Fraction

PlacentalextractSephacrylS-300Heparin-SepharoseAffinityColumn

Volume

90

360

10

3

Proteincone.mg/ml

112

11

8

0.1

totalmg

10080

3960

80

0.3

Activityspecificunits/mg

29

60

2150

281000

totalunits

29.2 X104

23.8X104

17.2X104

8.4X104

Yield %

100

82

59

29

PurificationFactor

1

i

74

9700

IRF activity was measured by gel-retardation assays after reduction with 2% /3-mercaptoethanol. 1 unit is defined as the amount of IRF which interactswith 3.5x 105 cpm of the high specific activity RNA transcribed from pSPT-fer. Taking into account that binding of IRF to this 46 base transcriptoccurs at a 1:1 ratio (10), I unit corresponds to about 3 ng of IRF.


54 Nucleic Acids Research

HEP FT Wi W2 W3 1 M KCl Elution HEP FT1 FT2

m w m

I1 2 3 4 5 6 7 8 9 10 11 12

Figure 3. Purification of IRF by affinity chromatography. Fractions generatedduring the procedure were analysed by the gel-retardation assay. Prepurified IRFeluted from a heparin column (lane 1) was adsorbed quantitatively on an affinitycolumn (Fig.l) with transcripts from pSPT-TR38 (containing 5 IREs). No IRFactivity was present in the flow-through (lane 2) or three subsequent washes (lanes3-5) . IRF was entirely recovered in the first 3 elutions with 1 ml 1 M KCl in25 mM Hepes buffer pH 7.5 (lanes 6-9) . The specificity of IRF binding to IREsin the affinity matrix was verified by incubating a heparin fraction first with aRNA affinity column lacking IREs (transcript from pSPT-TR35) and subsequentlyto the IRE-containing column (transcript from pSPT-TR38). The IRF activityin the first column flow-through (lane 11) was comparable to the one in the originalheparin fraction (lane 10), but no activity was present in the flow-through of thesecond column (lane 12).

PL S-300 HEP SEPH AFFINITY COLUMNkDa

- 205

— - 11697

— - 66

- - 45

8 M

Figure 4. Analysis by SDS/PAGE of IRF purification from human placenta.Proteins from the placental extract (lane 1), pooled IRF-positive S-300 fractions(lane 2), the heparin column flow-through (lane 3) and eluate (lane 4), as wellas the RNA affinity column flow-through (lane 5) and eluate (3, 2 or 1 /tg IRFin lanes 6—8) were analysed on a 7.5% polyacrylamide gel. All the samples werereduced with 100 mM dithiothreitol prior to SDS/PAGE. The gel was stainedwith Coomassie blue.

avoids any chemical derivatization of the RNA. To this end, weconstructed a vector that allows in vitro transcription ofpolyadenylated RNA. The RNA is then directly bound to poly(U)-Sepharose (Fig.l). Although the adsorbtion of RNA to theclassically used oligo(dT)-cellulose might be equally effective wepreferred to use poly(U)-Sepharose since it binds polyadenylatedRNA more firmly at low salt concentrations, a condition that iscritical to preserve the RNA-protein interaction. Two distinctaffinity columns were prepared, one with RNA containing the5 palindromic IREs from the 3' UTR of human TR mRNA, anda second control column with approximately 1250 bases of anRNA without IREs. In both cases, the RNA was bound to thepoly(U)-Sepharose prior to the incubation with protein fractionsso as to avoid a prolonged exposure of the affinity columns tothe possible presence of ribonucleases. Titration of the bindingcapacity using trace-labelled in vitro transcripts revealed that 1ml poly(U)-Sepharose beads are saturated at approximately 2nmol of RNA.

Both the binding of RNA to poly(U)-tails (not shown) andsubsequent interaction of IRF with the IRE-containing RNA(Fig.3) were found to be rapid and virtually complete within 3rounds of recycling on a small column at room temperature. NoIRF was retained on the control column (Fig.3, lane 11),however. In order to obtain a good recovery of IRF, it was criticalto reduce IRF with 2% /3-mercaptoethanol just prior to itsadsorbtion on the affinity column since the protein tends to oxidizespontaneously and thereby looses its RNA-binding property (21).We also noticed that the yield of IRF is dependent on the presenceof a ribonuclease inhibitor. The recovery of IRF by elution with1 M KCl from the affinity column was about 50% of the heparinfraction (Table 1). The overall yield in the purification procedureis close to 30% as determined by careful titration of IRF bindingactivity in gel-retardation assays with a short IRE-containingRNA. From one fifth of a placenta we obtain an IRF preparationwith about 300 /ig protein (Table 1). Based on the units of IRFrecovered, we estimate that more than 240 /tg of the preparationis able to bind to an IRE, indicating that the purity of IRF isat least 80%. A 10,000-fold purification in only a few steps seemshighly satisfactory. We are currently testing whether the gelfiltration step can be omitted. This might even further simplify

S-300 * dainkfc P-ma

S-300 ^ -me Inacthrabon reactivation

IRF + cSamida p-ma

IRF f i me jnadivaion teacSvalion

, 2 3 4 5 6

a*-

1 2 3 4 5 6

Figure 5. Activation of IRF in vitro. Human placental IRF fractionated bySephacryl S-300 (A) or purified by affinity chromatography (B) was analysedby the gel-retardation assay either after inactivation with azodicarboxylic acidbis[dimethylamide], a thiol-oxidizing diamide, or reactivation by 2% /3-mercaptoethanol. Lane 1 shows a control RNA bandshift with untreated IRF;lane 2, after reduction of IRF with 2% /3-mercaptoethanol; lanes 3 and 4: afteroxidation of IRF with 1.5 mM or 100 mM diamide, respectively; lanes 5 and6: after reduction with 2% /3-mercaptoethanol of IRF samples shown in lanes3 and 4.

the procedure of purifying specific RNA-binding protein;;. Whilewe were preparing the present manuscript two other groups havesucceeded in purifying IRF from liver (15,22). One group (15)has used a relatively complicated classical procedure, whereasin the other study (22) IRF was isolated by the biotin-agaroseaffinity method. For reasons that we are not able to judge,however, the recovery using this latter method was no more than0.2%. Possible difficulties encountered in protein purificationusing an RNA affinity column may be related to the trimmingof RNA by ribonucleases. This could lead to an incompleteretention of RNA-protein complexes on the solid support. Wehave monitored the leakiness of our affinity column by measuringthe release of 32P-labelled RNA during the protein binding,washing and elution steps. About 15 % were released in total,mainly during the protein binding step (data not shown). Thiscould account for a 30 % loss of IRF during the purification.

Biochemical properties of IRFTo assess the purification of IRF, the protein fractions from eachstage of the isolation procedure were analysed by SDS/PAGE(Fig.4). The final fraction from the affinity column revealed two


Nucleic Acids Research 55

major protein bands corresponding to molecular weights of 95and 100 kDa and a minor species of 47 kDa. No such proteinswere visible in the eluate from the control column (result notshown). Several lines of evidence indicate that the 95/100 kDadoublet corresponds to IRF. The molecular weight agrees bothwith the migration of IRF in gel filtration (Fig.2 and ref. 10) andwith our previous UV crosslinking experiments which hadidentified two IRE-IRF complexes only slightly above the sizesreported here (10). Moreover, compatible with the purity of IRFestimated by activity measurements (Table 1), the two bandsaccount for more than 80% of the Coomassie-blue stainedproteins in the affinity purified fraction. We have no indicationspresently about the nature of the minor 47 kDa component.Although this protein has not been observed in previous RNA-protein crosslinking experiments (10) and does not seem to bepresent in stoichiometric amounts compared to IRF, we observedthat it copurifies specifically with IRF. Preliminary gel filtrationexperiments using FPLC and the affinity purified fraction suggestthat the protein is physically associated with IRF (unpublishedobservation).

Placental IRF purified in the present study closely resemblesIRF from liver whose size has recently been reported to be about90 kDa (9,15,22). Yet there is a difference. While liver IRFmigrates as a single species in SDS/PAGE (15,22), the presentIRF prepared from placenta is a doublet. It is not clear whetherthis discrepancy is due to differences among tissues, or whetherproducts from two genes, allelic forms or a post-translationalmodification are the cause of the observed heterogeneity. Wedetermined that the two forms of human placental IRF have verysimilar pi of 6.45 and 6.55 (data not shown), which suggeststhat they are highly related. It should also be noted that the twoforms of IRF are present in nearly equimolar amounts. We foundno evidence for preferential in vitro binding of either species toIREs from ferritin or transferrin receptor mRNA (data notshown), nor was the doublet related to the state of IRF reduction.

Probably the most intriguing question about IRF concerns theactivation of its RNA-binding properties by cellular ironchelation. Although the mechanism of this process remains largelyunknown, recent evidence indicates that IRF activation involvesthe reduction of a sensitive disulfide bridge inducing presumablya conformational change (21). Using the purified placentalpreparation we confirm that in vitro activation of IRF dependson its reduction (Fig.5). Oxidation of the protein by 1.5 mMdiamide, a thiol-oxidizing agent specific for intracellularglutathione (29), abolished entirely the binding of IRF to an IRE-containing transcript, and the activity was restored by theexposure to 2 % /3-mercaptoethanol. A fraction of IRF obtainedon Superose 12 by FPLC, which lacks the 47 kDa protein hadthe same properties (not shown). Thus, based on these results,it appears that IRF from placenta is activated in a similar fashionto IRF from human cell lines. This suggests that the regulatoryfactor is involved in the iron-metabolism of different tissues. Thepossibility of isolating IRF in large quantities permits to envisageits sequencing and cloning as well as a detailed analysis of thephysical interaction of IRF with RNA. With respect to the presentaffinity purification approach, we are confident that it has thepotential of being generally applicable to the isolation of specificRNA-binding proteins.

The present work was supported by the Swiss National ScienceFoundation, the Swiss Ligue for Cancer Research and the ForesFoundation.

REFERENCES

1. Zahringer, J., Baliga, B.S. and Munro, H.N. (1976) Proc. Natl. Acad. Sci.USA 73, 857-861.

2. Aziz, N. and Munro, H.N. (1986) Nucl. Acids Res. 14, 915-927.3. Rogers, J. and Munro, H. (1987) Proc. Nail. Acad. Sci. USA 84, 2277-2281.4. Aziz, N. and Munro, H.N. (1987) Proc. Natl. Acad. Sci. USA 84,

8478-8482.5. Hentze, M.W., Rouault, T.A., Caughman, S.W., Dancis, A., Harford, J.B.

and Klausner, R.D. (1987) Proc. Natl. Acad. Sci. USA 84, 6730-6734.6. Owen, D. and Kuhn, L.C. (1987) EMBO J. 6, 1287-1293.7. Milliner, E.W. and Kuhn, L.C. (1988) Cell 53, 815-825.8. Casey, J.L., Di Jeso, B., Rao, K., Klausner, R.D. and Harford, J.B. (1988)

Proc. Natl. Acad. Sci. USA 85, 1787-1791.9. Leibold, E.A. and Munro, H.N. (1988) Proc. Natl. Acad. Sci. USA 85,

2171-2175.10. Mullner, E.W., Neupert, B. and Kuhn, L.C. (1989) Cell 58, 373-382.11. Koeller, D.M., Casey, J.L., Hentze, M.W., Gerhardt, E.M., Chan, L -

N., Klausner, R.D. and Harford, J.B. (1989) Proc. Natl. Acad. Sci. USA86, 3574-3578.

12. Rouault, T.A., Hentze, M.W., Caughman, S.W., Harford, J.B. and Klausner,R.D. (1988) Science 241, 1207-1210.

13. Walden, W.E., Daniels-McQueen, S., Brown, P.H., Gaffield, L., Russell,D.A., Bielser, D., Bailey, L.C. and Thach, R.E. (1988) Proc. Natl. Acad.Sci. USA 85, 9503-9507.

14. Brown, P.H., Daniels-McQueen, S., Walden, W.E., Patino, M.M., Gaffield,L., Bielser, D. and Thach, R.E. (1989)/ Biol. Chem. 1KA, 13383-13386.

15. Walden, W.E., Patino, M.M. and Gaffield, L. (1989)7. Biol. Chem. 264,13765-13769.

16. Casey, J.L., Hentze, M.W., Koeller, D.M., Caughman, S.W., Rouault,T.A., Klausner, R.D. and Harford, J.B. (1988) Science 240, 924-928.

17. Chan, L.-N.L., Grammatikakis, N., Banks, J.M. and Gerhardt, E.M. (1989)Nucl. Acids Res. 17, 3763-3771.

18. Hentze, M.W., Caughman, S.W., Casey, J.L., Koeller, D.M., Rouault,T.A., Harford, J.B. and Klausner, R.D. (1988) Gene 72, 201-208.

19. Ward, J.H., Jordan, I., Kushner, J.P. and Kaplan, J. (1984) J. Biol. Chem.259, 13235-13240.

20. Rouault, T., Rao, K., Harford, J., Mania, E. and Klausner, R.D. (1985)J. Biol. Chem. 260, 14862-14866.

21. Hentze, M.W., Rouault, T.A., Harford, J.B. and Klausner, R.D. (1989)Science 244, 357-359.

22. Rouault, T.A., Hentze, M.W., Haile, D.J., Harford, J.B. and Klausner,R.D. (1989) Proc. Natl. Acad. Sci. USA 86, 5768-5772.

23. Kuhn, L .C , McClelland, A. and Ruddle, F.H. (1984) Cell 37, 95-103.24. Costanzo, F., Colombo, M., Staempfli, S., Santoro, C , Marone, M., Frank,

R., Delius, H. and Cortese, R. (1986) Nucl. Acids Res. 14, 721-736.25. Schneider, C , Owen, M.J., Banville, D. and Williams, J.G. (1984) Nature

311, 675-678.26. Okayama, H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289.27. Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and

Green, M.R. (1984) Nucl. Acids Res. 12, 7035-7056.28. Konarska, M.M. and Sharp, P.A. (1986) Cell 46, 845-855.29. Kosower, N.S., Kosower, E.M., Wertheim, B. and Correa, W.S. (1969)

Biochem. Biophys. Res. Common. 37, 593-596.30. O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021.31. Grabowski, P.J. and Sharp, P.A. (1986) Science 233, 1294-1299.32. Ruby, S.W. and Abelson, J. (1988) Science 242, 1028-1035.

ACKNOWLEDGEMENTS

We wish to thank Dr. Ernst Mullner for stimulating discussionsand Mrs C. Ravussin for assistance in preparing the manuscript.


a high yield affinity purification method for specific rna- binding

Documents