A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA

Cell, Vol. 58, 373-382,

July 28, 1989, Copyright

0 1989 by Cell Press

A Specific mRNA Binding Factor Regulates Iron-Dependent Stability of Cytoplasmic Transfekin Receptor mRNA Ernst W. Miillner, Barbara Neupert, and Lukas C. Kuhn Swiss Institute for Experimental Cancer Genetics Unit CH-1066 Epalinges Switzerland

Research

Iron regulates human transferrin receptor (MR) expression by modulating the stability of cytoplasmic hTR mRNA. This regulation requires a distinct secondary structure in the mRNA 3’ untranslated region. We identified a specific cytoplasmic factor that binds simultaneously to four homologous palindromes within the regulatory domain. Iron chelator induced the RNA binding activity 25-fold in parallel with mRNA. Upon the addition of iron salts, a rapid decay of factor activity closely preceded MR mRNA degradation, indicating a causal relation. Induction and decay occurred posttranscriptionally. Binding of the factor to MR mRNA palindromes was competed by 5’ regulatory sequences of ferritin mRNA, which are responsible for iron-dependent translational control. These results suggest that cellular iron maintains its homeostasis by coordinate regulation of MR and ferritin expression via a common factor. Introduction Progress made in recent years toward the understanding of regulatory mechanisms involved in eukaryotic gene expression has mainly focused on factors binding to promoter and upstream regulatory elements (for review, see Ptashne, 1988). It is clear, however, that posttranscriptional control of RNA processing and turnover contributes to the maintenance of cytoplasmic mRNA steady-state levels. Subsequent to translocation into the cytoplasm, mRNAs are subject to dynamic interactions with specific proteins that may determine their subcellular localization, translational control, or half-lives (reviewed by Richter, 1988). The stability of a given mRNA can be modified in response to a change of physiological conditions. For example, creatine phosphokinase mRNA is selectively stabilized by insulin in differentiating rat myoblasts (Pontecorvi et al., 1988) the half-life of mRNA for the heat shock protein hsp70 increases around lo-fold after heat shock in HeLa cells (Theodorakis and Morimoto, 1987) and epidermal growth factor greatly increases the stability of the mRNA for its own receptor in human carcinoma cells (Jinno et al., 1988). In most cases the molecular basis for these differential stabilities is unknown, with the notable exception of histone and tubulin mRNA. Histone mRNAs have characteristic 3’terminal structures that are involved in cell cycle-specific degradation coupled to translation (for review, see Schtimperli, 1988). Tubulin mRNA gets destabi-

the

lized as a consequence of cotranslational binding of mature tubulin to the nascent polypeptide chain (reviewed by Cleveland, 1988). We have previously shown that the stability of human transferrin receptor (MR) mRNA is regulated by intracellular levels of iron (Miillner and Kuhn, 1988). When cells are treated with the iron chelator desferrioxamine, cytoplasmic hTR mRNA becomes 20-fold more stable. Full induction requires about 15 hr. After the addition of iron salts, MR mRNA decays with a half-life of 1.5 hr. The iron chelator produces no significant effect on MR gene transcription nor on the nuclear processing of primary transcripts. By transfecting mouse L cells with MR cDNA (Kuhn et al., 1984) and derived plasmid constructions, we found that iron-dependent regulation requires the presence of the 3 untranslated region (3’ UTR) (Owen and Kuhn, 1987). Analysis of a series of deletion mutants identified two domains in the 3’ UTR of hTR mRNA that form a specific secondary structure (MulIner and Kuhn, 1988). This regulatory domain contains a prominent stem-loop of about 60 bases and five repeats of a palindromic sequence element. Deletion of more than one of the palindromes, as well as point mutations in the stem-loop, completely abolished the iron-dependent regulation of hTR transcripts. The effect of iron on MR mRNA stability raised the question as to whether the regulatory region is the target for a specific iron-dependent RNAase or for binding of a cytoplasmic protein. To identify and characterize such a factor in extracts of mouse and human cells, we used a gel-retardation technique to analyze RNA-protein complexes (Konarska and Sharp, 1986). We found proteins that bind with high specificity to palindromes in the MR mRNA 3’UTR. Binding activity is inversely correlated with the level of intracellular iron. The kinetics of induction and the location of binding sites strongly suggest a direct role of the factor in stabilizing hTR mRNA. The molecular weight and binding specificity indicate that the factor is related to the one controlling translation of mRNA for the iron storage protein ferritin (Leibold and Munro, 1988). Results Binding of an Iron-Dependent Cytoplasmic Factor to the MR mRNA 3’ UTR To investigate in vitro whether the iron-dependent regulation of MR mRNA stability involves the binding of a specific cytoplasmic factor to the regulatory elements in the 3’ UTR (Owen and Kuhn, 1987; Miillner and Kuhn, 1988), we used a method for the analysis of RNA-protein complexes by nondenaturing gel electrophoresis (Konarska and Sharp, 1986). Cytoplasmic protein extracts were prepared from logarithmically growing mouse L cells and incubated with a =P-labeled 2.3 kb transcript corresponding to the entire 3’ UTR of MR mRNA. To remove unspeciftc RNA-protein complexes, the samples were digested with RNAase T, and subsequently incubated with heparin (Leibold and Munro, 1988). Only RNA fragments

Cdl 374

pretreatment none ng competitor hTR

of cells Fe3+

Desf

none

RNA P-actin

Figure 1. Specific Complexes between RNA from the MR 3’ UTR and Cytoplasmic Protein Radiolabeled RNA (0.1 ng. 1.3 x 10s dpmltrg) transcribed in vitro from pSPTTRl1, containing the 2.3 kb of the MR 3’ UTR, was incubated at room temperature with 50 rg of cytoplasmic protein extracts from mouse L cells. The extracts were from untreated cells, cells grown for 30 hr in the presence of 50 uM desferrioxamine, or 20 uglml of ferric ammonium citrate. RNA-protein binding was competed by various amounts of unlabeled RNA from pSPT-TRll or a 1.1 kb mouse p-actin RNA transcribed from pSPT-act. After sequential addition of RNAase T, and heparin, RNA-protein complexes were analyzed in a 4% nondenaturing polyacrylamide gel.

that are tightly associated with protein remain protected from RNAase digestion and are resolved by nondenaturing polyacrylamide gel electrophoresis. The experiments clearly revealed the formation of a complex between a cytoplasmic factor and the MR mRNA 3’UTR (Figure 1). The binding of the factor was specific since it could be competed by the homologous unlabeled MR RNA but not by unlabeled f3-actin RNA (Figure 1). Similarly, no competition was observed with antisense RNA from the 3’ UTR of MR mRNA or single-stranded DNA corresponding to the same region (data not shown). By competition with different amounts of MR RNA, it could be estimated that 50 ug of protein from a cytoplasmic L cell extract contains enough binding factor to saturate more than 1 ng of the 2.3 kb input RNA. Based on similar experiments with shorter transcripts (see below), and taking into consideration the amount of 32P-RNA protected in the RNA-protein complexes, we calculated that the factor is present in approximately 5000 copies per cell. Using an extract from cells cultured for 30 hr in the presence of the iron chelator desferrioxamine, we observed a more than lO-fold increase in binding activity (Figure 1). Pretreatment of the cells with ferric ammonium citrate did not alter the amount of RNA binding factor. No significant free binding activity was found in nuclear extracts or polysomal preparations (data not shown). These findings indicate the existence of a factor inducible by iron chelators in the cytoplasm. Because binding of the factor occurred within the 3’ UTR known to be necessary for irondependent destabilization of MR mRNA (Miillner and Kuhn, 1988), a direct correlation between the two phenomena may be inferred.

The Factor Binds at Specific Sites within the Regulatory Domain of MR mRNA By analysis of deletion mutants, we have previously defined the precise location of regulatory sequences involved in iron-dependent hTR mRNA stability (Miillner and Kiihn, 1988). Our original computer calculations (Zuker and Stiegler, 1981) predicted that the regulatory region is probably folded into an extended secondary structure with a stem-loop and paired direct repeats (Figure 2, structure b). The sequence of these repeats is highly palindromic. The regulated phenotype correlated with the presence of the stem-loop, although flanking sequences between positions 3847-3970 containing the palindromic repeats were also required. Recent modifications in the thermodynamic constants for RNA base pairing (Freier et al., 1988) led us to recalculate the secondary structure. Using these new parameters, the program predicted that the palindromic direct repeats within the regulatory region would fold as five hairpins rather than base pairing with each other (Figure 2, structure a). In considering a possible role of the binding factor in iron-dependent regulation of MR mRNA expression, it was of interest to determine whether the binding sites on MR RNA would correlate with structures in the regulatory domain. Different regions of the 3’ UTR were subcloned into the phage T7 promoter-containing transcription vectors pSPTW19 to produce either radioactive probes or unlabeled competitor RNAs (Figure 2). The RNA-protein complexes observed with a probe covering only the regulatory domain (pSPT-TR21) were indistinguishable from those obtained with a probe containing most of the MR mRNA 3’UTR (pSPT-TRll). No binding was observed with probes mapping 5’ to position 3342 or 3’ to position 4048, nor did RNA from the central fragment between nucleotides 351 l-3878 (pSPT-TR22) bind a factor (data not shown) or act as a competitor in our assay (Figure 2). This indicated that the protein binds exclusively to the regulatory region of MR mRNA 3’ UTR. RNA from pSPT-TRPl completely competed the binding of factor to transcripts from pSPT-TR11 and was used as a probe in the following experiments. Competition with RNAs from various parts of the regulatory region showed that the binding was limited to sequences containing one or more palindromes (Figure 2). No competition by the stem-loop was observed with the transcripts from pSPT-TR27 and pSPTTR28. This finding is of particular interest since several deletion and point mutants have proven the importance of this structural element for iron-dependent MR expression (Miillner and Kuhn, 1988). In conclusion, it seems that the palindromes represent the binding structure for the cytoplasmic factor. This was confirmed by the complete competition with RNA from pSPT-TR34, which has only a single palindrome. Similarly, transcripts from pSF’T-TR28 and pSPTTR31 were good competitors, indicating that palindromic sequences in different parts of the regulatory domain are functionally equivalent in binding the cytoplasmic protein. However, we observed that an RNA containing only the first palindrome (pSPT-TRll cut at position 3499) did not bind the factor measurably and was 20 times less effective as a competitor (data not shown).

Regulation 375

of Transferrin

Receptor

mRNA

Stability

a

competition

plasmid

pSPT.TW, pSPT-TR21

+ + +

pSPT-TR11 @PT.TR12 pSPT-TR21 pSPT-TR22

+ + + +

pSPT-TR23 pSPT-TRZ4 pSPT-TR25 pSPT-TR26 pSPT-TR27 pSPT-TR28 pSPT-TR29 pSWTR31 pSPT-TR32 @PI-TP33 pSPT

Figure

TM4

2. Determination

of Protein

Binding

Sites in the Regulatory

Region

of hTR mRNA

The regulatory region required for iron-dependent stability of MR mRNA is represented in the secondary structure models (a) and (b). They contain five palindromes (open boxes), a stem-loop structure (light grey shaded box), and two perfect inverted repeats (dark grey shaded box). RNAs transcribed in vitro from plasmids containing various parts of the MR mRNA regulatory domain were analyzed for their ability to compete the binding of cytoplasmic factor to hTR RNA. Binding reactions were carried out as described in Experimental Procedures with labeled RNAs from pSPTTRl1 or pSPT-TR21 (broken lines) in the presence of a lOO-fold molar excess of different competitor RNAs (solid lines). Complete competition is indicated by a + sign and shown in the insert.

Multiple Binding of the Cytoplasmic Factor to the Palindromes in MR mRNA To test further whether each of the palindromes was able to bind a cytoplasmic factor, we decided to determine the molecular weight of RNA-protein complexes by gel filtration. The size of the unbound factor was measured in cyto-

plasmic extracts from human placenta or mouse L cells. The extracts were separated on a Superose 6 column by FPLC and each fraction was analyzed for binding activity. We identified a single peak of activity migrating at a size corresponding to 105 kd in both human and mouse extracts (Table 1). To determine the respective sizes of

Table

between

Extract

1. Molecular

from

Weight

Determination

RNA from

Mouse pSPT-TR34 pSPT-TR32 pSPT-TR24 pSPT-TR21 Human

by FPLC

Palindromes

of Complexes

Formed

M, of Complex

(kd)

hTR

RNA and Mouse M, of RNA Component

-

-

-

1 2 3 5

145 330 500 545

27 63 137 145

-

-

-

-

pSPT-TR34 pSPT-TRPI

1 5

135 615

27 145

(kd)

or Human

Cytoplasmic

Factor

M, of Protein Component (kd) 105 118 267 363 400 105 108 470

The molecular weight of unbound factor was estimated by fractionation of protein extracts on Superose 6 by FPLC. The RNA binding activity in the factions was determined by the standard assay method (see Experimental Procedures). To measure the size of RNA-protein complexes, 50 ug of cytoplasmic protein was incubated with 0.5 ng of the different labeled RNAs as indicated and 200 ug of E. coli rRNA. Before loading the 290 pl sample onto the column, heparin was added to a final concentration of 5 mglml. The digestion with RNAase T, was omitted. The peak of radioactivity from RNA-protein complexes was correlated to the molecular weights of protein standards (thryglobulin, 669 kd; catalase. 232 kd; transferrin, 80 kd).

A

A c

pSPT-TR34

0

complexes correlated well with the number of palindromic sequences in each RNA, except for the one from pSPTTR21, which seemed to have only four binding sites (Table 1). RNA transcribed from pSPT-TR22 (Figure 2) which contains no palindromes, did not shift to a higher molecular weight, indicating that it has no binding sites for any cytoplasmic protein. The experiments demonstrate that at least four of the palindromic sequences in MR mRNA can bind the cytoplasmic factor concurrently.

” 0 ”

A A

pSPT.

: G

probe RNA hTR protein human

(pSPT-fer)

extfact

ng competitor 0 :: 0~~,-0~.-~0-,-~Orv-~

1

ferritin

(pSPT-TR34)

23456789

mouse

human

rmuae

hTR RNA 0

0 0

10

a

0 0

11

12

13

14

0

z

15

16

Figure 3. Cross-Competition between Palindromes of MR and Ferritin mRNA for Binding of Cytoplasmic Proteins (A) The sequence and predicted secondary structure of transcripts from pSPT-TR34, corresponding to the fifth palindrome in the regulatory region of hTR mFtNA, and pSPT-fer containing the iron regulatory element of human ferritin heavy chain mRNA are shown. The s% quences are numbered according to Schneider et al. (1964) for hTR, and Costanzo et al. (1966) for ferritin mRNA, respectively. The part of the RNA transcribed from pSPT vector sequences is indicated by lower case letters. (B) Labeled RNA (0.01 ng) from pSPTTR34 (lanes l-6) or pSPT-fer (lanes 9-16) was incubated with 50 ug of extracts from mouse L cells (lanes 5-6 and 13-16) or human placenta (lanes l-4 and 9-12) in the presence of various amounts of unlabeled pSPT-TR34 competitor RNA as indicated. The RNA-protein complexes were analyzed as described in the Experimental Procedures.

RNA-protein complexes, we incubated limiting amounts of labeled transcripts, which have different numbers of MR palindromes, with either mouse or human extract and separated the complexes on Superose 6. RNAase T1 digestion was omitted in these experiments to observe the full size of the RNA-protein complexes. A shift in the apparent molecular weight of the radioactive RNA indicated the formation of RNA-protein complexes. The sizes of the

Regulatory Elements in MR and Ferritin mRNA An? Recognized by the Same Factor Iron regulates not only the abundance of MR mRNA. At high iron concentrations, a pool of free mRNA for the iron storage protein ferritin is mobilized onto polysomes (Aziz and Munro, 1986; Rogers and Munro, 1987). A potential hairpin structure in the 5’ untranslated region of ferritin mRNA is responsible for this translational control (Aziz and Munro, 1987; Hentze et al., 1987) and can specifically bind a cytoplasmic protein (Leibold and Munro, 1988). Recently, Casey et al. (1988) have shown that a single MR palindrome is also sufficient to confer iron-dependent translational control to human growth hormone mRNA when introduced into the 5’ UTR. This led us to test whether the proteins binding to MR mRNA3’UTR and ferritin mRNA 5’ UTR are identical. A radiolabeled RNA transcribed from a short DNA sequence corresponding to the iron regulatory element of human ferritin heavy chain (pSPT-fer) was used as a probe in binding assays with human and mouse cytoplasmic extracts. The reaction was competed with different concentrations of unlabeled RNA from the MR 3’ UTR (pSPTTR11). Labeled RNA from pSPTTR34 containing a single MR palindrome was analyzed in parallel as a control (Figure 3). Two distinct complexes formed when ferritin RNA was incubated with an extract from mouse L cells, and a single complex formed with a human placental extract. They migrated at the same position as those seen with the MR probe. The formation of complexes between labeled ferritin RNA and protein could be completely abolished by adding a lOO-fold molar excess of cold MR RNA. The same amount of unlabeled competitor was required to compete binding to the control MR probe. In a reverse experiment, we were able to inhibit the interaction of the cytoplasmic factor with labeled MR RNA by unlabeled ferritin RNA (data not shown). The results indicated that the factor recognizes both types of iron regulatory elements. For this reason, we propose to name this cytoplasmic protein “iron regulatory factor” (IRF). To investigate whether IRF binding to MR and ferritin mRNAs is identical, complexes between protein and labeled RNAs from pSPWR34 or pSPT-fer were digested with RNAase T,, covalently linked by UV irradiation, and resolved on SDS-polyacrylamide gels under reducing conditions (Figure 4; Leibold and Munro, 1988). Both the MR and ferritin probes specifically labeled mouse cytoplasmic proteins that migrated at 97 kd and 103 kd. The affinity labeling could be competed with an excess of unlabeled RNA from either pSPT-TR34 or pSPT-fer. By measuring the radioactivity of protected versus input RNA, we

Regulation 377

of Transferrin

Receptor

probe RNA hTR

mRNA

Stability

control

hTR ferritin

+Desf

competitor

-Desf

+Desf

-Desf

-97 -80 -67

123456 Figure 4. UV Cross-Linking and SDS-PACE MR and Ferritin RNA Regulatory Elements

of Mouse

IRF Bound to

Labeled transcripts of the fifth palindrome of MR 3’ UTR (pSPT-TR34; lanes l-3) or the iron regulatory element of ferritin 5’ UTR (pSPT-fer; lanes4-6)were used in RNA-protein binding reactions followed by UV cross-linking. Protein extracts were isolated from mouse L cells. Binding specificity was assessed by competition with a IOO-fold molar excess of either unlabeled pSPTTR34 RNA (lanes 2 and 5) or pSPT-fer RNA (lanes 3 and 6) during complex formation. The cross-linked RNA-protein complexes were analyzed on a 10% SDS-polyacrylamide gel under reducing conditions. The migration of molecular weight markers is indicated.

determined that the RNA protected against RNAase T, has an average length of 42 bases. Its contribution to the apparent molecular weight of the complexes is therefore about 14 kd. Accordingly, the mass of the cross-linked proteins is 83 kd and 89 kd. A rat liver protein with a molecular weight of 87 kd that binds to the iron regulatory element of rat H- and L-ferritin mRNA has been recently reported (Leibold and Munro, 1988). The reason for the appearance of a double band in our UV cross-linking experiments is at present unknown. UV cross-linking of either RNA probe with human placental extract also yielded a double band with molecular weights of 97 kd and 103 kd (data not shown). IRF Does Not Prevent hTR mRNA Binding to Polysomes The 5’ regulatory element of ferritin mRNA has been shown to be required for iron-dependent translational control (Aziz and Munro, 1987; Hentze et al., 1987). At low iron concentrations, IRF binds to the regulatory element. This observation correlates with the existence of a pool of free ferritin mRNA, suggesting that IRF prevents association of the mRNA with polysomes (Rogers and Munro, 1987). At high iron levels, IRF does not bind and the free ferritin mRNA pool is mobilized onto polysomes and translated. This raised the possibility that IRF influences hTR mRNA distribution between a free and polysome-bound mRNA pool. We measured this distribution in Ltk- cells transfected with a full-length hTR cDNA clone. Cells were grown either in the presence or absence of iron chelator prior to prepa-

+B Figure

5. Polysome

Fractionation

of Cytoplasmic

WR mRNA

The distribution of hTR mRNA on polysomes was measured in mouse L cells transfected with the regulated MR cDNA clone pTR-61 (Mgllner and Kiihn, 1966). Cytosolic extracts from cells grown for 30 hr in the presence or absence of desferrioxamine were separated on 15%-40% linear sucrose gradients and collected in 16 fractions as described in Experimental Procedures. The RNA from each fraction was transferred to nylon filters and hybridized with either 32P-labeled DNA fragment from MA cDNA or 92-microglobulin cDNA. The top (T) and bottom (B) of the gradients are indicated by arrows.

ration of cytoplasmic extract. The samples were loaded onto linear 150/b-40% sucrose gradients that separate polyribosomes, monosomes, ribosomal subunits, and postribosomal supernatant containing free mRNA. Fractions with equal volume were harvested. The identity and quality of RNA in each fraction were assessed by electrophoresis on nondenaturing agarose gels and ethidium-bromide staining (data not shown). Desferrioxamine did not influence the stability and overall distribution of the major RNA species. The RNA from each fraction was blotted and hybridized to ZP-labeled cDNA probes specific for MR or f32-microglobulin, whose mRNA is not influenced by iron levels. It was found that, in vivo, all detectable MR mRNA is associated with polysomes (Figure 5). The lack of free MR mRNA could not be caused by extensive RNA degradation since the 58microglobulin probe detected a pool of mRNA that was not associated with polysomes. Although the total amount of MR mRNA was increased under the influence of desferrioxamine, there was no effect on its relative distribution throughout the gradient. These findings suggest that stabilization of MR mRNA by desferrioxamine is not achieved by redistribution of free versus polysome-bound mRNA. Therefore, the effect of IRF on hTR mRNA must be different from the effect responsible for translational control of ferritin mRNA.

Cell 370

0

2

4

time after desf

6

6

10

addition

(hours)

12

but it was delayed 8 hr by the translation inhibitor cycloheximide. These results demonstrate a posttranscriptional mechanism of factor activation, possibly requiring protein synthesis. An independent control experiment using pulse labeling with [SSJmethionine showed that translation inhibition by cycloheximide was only effective for 8 hr (data not shown). Replacement of iron chelator in induced cultures by ferric ammonium citrate causes a rapid decay of MR mRNA with a half-life of 1.5 hr (Miillner and Kuhn, 1988). Under the same conditions, the RNA binding activity decreased even faster and was reduced by 50% within 30 min (Figure 86). A level of RNA binding activity close to the one of untreated cells was reached within 2 hr. Actinomycin D and cycloheximide did not significantly affect this rate of inactivation. Thus, the time courses of activation and inactivation of the RNA binding factor correlate well with the iron-dependent accumulation and decay of MR mRNA. Discussion

o

2 time

4 after desf

6 removal

8

10

12

(hours)

Figure 6. Time Course of Iron-Dependent Induction and Loss of IRF Binding Activity Levels of IRF binding activity were measured in extracts from cells cultured for various lengths of time with 50 uM desferrioxamine alone (circles), or in the presence of an additional 5 uM actinomycin D (open triangles), or 40 uM cycloheximide (squares) (A) Alternatively, cells were cultured for 24 hr with desferrioxamine and then were given fresh medium containing iron salts (circles), with additional actinomycin D (open triangles) or cycloheximide (squares) (B) Extracts from each time point were titrated for IRF activity by incubating 50 ug of cytoplasmic protein with 0.5 ng of labeled pSPTWt21 RNA in the presence of 0 ng, 5 ng, or 50 ng of homologous unlabeled RNA. RNA-protein complexes were separated in nondenaturing gels. The amount of =P-RNA protected by bound IRF against RNAase T, was quantitated by densitometry of autoradiographs.

IRF Activity Is Induced Fbsttranscriptionally From the preceding results, the question arises as to whether the binding of IRF stabilizes MR mRNA. If this were the case, IRF activity should be induced by the iron chelator with kinetics that are similar to the rate of MR mRNA accumulation. We have previously shown that the addition of desferrioxamine to cell cultures increases cytoplasmic MR mRNA 30-fold within 15 hr (Miillner and Kuhn, 1988). Therefore, the RNA binding activity was quantitated in L cell extracts prepared at different time points after the addition of desferrioxamine. Following a lag period of 2 hr, the amount of IRF activity increased steadily and reached a 25-fold higher level 12 hr after the addition of chelator (Figure 8A). This induction could not be blocked by the transcription inhibitor actinomycin D,

The purpose of the present study was to identify a transacting factor responsible for iron-dependent regulation of cytoplasmic MR mRNA stability. By incubating in vitro-labeled RNA transcripts with cytoplasmic extracts from mouse L cells or human placenta, we were able to find an RNA binding protein that interacts with the 3’ UTR of the mRNA. Two lines of evidence argue in favor of a direct role of this factor in MR mRNA stability. First, the activity of this factor, which we call IRF, was itself regulated by iron, and second, its binding sites on MR mRNA coincided with palindromic elements that are required for regulation. These results strongly suggest that IRF acts as an inhibitor for the degradation of MR mRNA. Desferrioxamine increased the pool of free cytoplasmic factor 25fold over a 12 hr period, with a rate that paralleled perfectly the accumulation of MR mRNA. The induction of factor activity required translation but not transcription, suggesting that IRF itself or a cofactor has to be synthesized de novo. We propose that IRF binds rapidly to available palindromes on newly synthesized hTR mRNA, thereby stabilizing these transcripts. Upon removal of iron chelator from the medium, rapid inactivation of IRF preceded degradation of MR mRNA. This would be expected for a causal relationship between IRF binding and RNA stability. Our present results show that IRF inactivation is not inhibited by cycloheximide or actinomycin D. In contrast, we have previously observed that upon iron addition to cells, hTR mRNA degradation was prevented by these compounds (MulIner and Kuhn, 1988). This indicates that mRNA turnover must be influenced by a second component, which is regulated differently than IRF. Consistent with this notion is our previous detection of a stem-loop structure in the regulatory domain of MR mRNA that was absolutely required for iron-dependent control of hTR expression. IRF does not bind to this sequence element. We propose that the stem-loop is the substrate for an RNAase that is sterically hindered by the binding of several IRF molecules to the adjacent palindromes (Figure 7). In accordance with this, the actual endonucleolytic cleav-

Regulation 379

of Transferrin

Receptor

mRNA

Stability

TRANSFERRIN RECEPTOR

lnhlbltlon of

FERRITIN

mRNA Degradation

Figure 7. Model for Coordinate Ferritin Expression

InhsbWm

Regulation

of

of Transferrin

Translation

Receptor

and

In addition to the mRNA and ribosomes, IRF is indicated in dark grey. The RNAase, postulated to attack the stem-loop in MR mRNA, is indicated as an open circle.

age would have to occur at a specific site, but independently of iron concentrations. Thus far, we have not been able to identify an RNAase that processes MR mRNA specifically. In several systems, as for example those of tubulin (reviewed by Cleveland, 1988) c-myc (Pei and Calame, 1988) or histones (for review, see Schtimperli, 1988) differential mRNA degradation is linked to protein synthesis. RNAases involved in these regulatory processes may thus be associated with ribosomes. This has already been demonstrated in the case of histone H4 mRNA degradation (Ross et al., 1987; Peltz et al., 1987). Because MR mRNA is found exclusively in the polysome fraction at both high and low iron levels (Figure 5) it seems likely that its degradation also occurs on ribosomes. In some

Table

2. Sequence

Palindromes

Comparison

of Iron Regulatory

Elements

in hTR and Ferritin

CAGAGU CAGUGC CAGUGU CAGUGA CAGUGU

= CUUCC CUUCC UCUCC UUCCC

AUAAAUGG AUAAUUAU AUAAUGUA AUAUGUUA AUAAUUUU

CAGUGU CAGUGC CAGUGU CAGUGC CAGUGU CAGUGC

= UUGAA UUGAC UUGGA UUGAA UUGGA

CGGAACAG CGGAACCC GAACAGAU CGGAACCC CGGAACCC CGGAACCG

mRNAs

in hTR 3’ UTR

4 UUAUUUAU AUAAUUAU CACAUUAU GUCUGUAU GUAAUUAU

C C C C C

Palindromes

in Ferritin

+ GUAUCUUG GUUUCCUG GUCUCUUG GUUUCCUG AGUUCUUG GGUUCCUG .

C C C C C C

l

AGUGA GGAAG GGGAG GGAGA GGGAA

(a)

W w 09 W

5’ UTR l

= UUCAA UUCAA UUCAA UUCAA GUCAA ‘

C

(9

* AA

NNNNNNNN

aspects, regulation of hTR mRNA turnover is nevertheless clearly distinct from the systems mentioned above. Histone mRNAs lacking a poly(A) tail are degraded by an exonucleolytic process. Moreover, histone and tubulin mRNA turnover are autocatalytic as free histone or tubulin protein increase the decay of their respective mRNA (Peltz and Ross, 1987; Yen et al., 1988). Such a feedback control is excluded for MR because no free polypeptide is present in the cytoplasm. To our knowledge, iron-dependent stabilization of MR mRNA by IRF represents one of the first cases where a cytoplasmic protein is shown to act as a repressor of mRNA degradation in higher eukaryotes. Unlike other RNA binding proteins, as those involved in 3’ end formation, splicing, or recognition of poly(A) tails and 5’ cap structures, IRF recognizes a sequence element highly specific for MR and ferritin mRNA. The sequence and structure requirements for binding of IRF to mRNA were evaluated by comparing the regulatory domains of MR and ferritin mRNAs from rat, human, bullfrog, and chicken (Table 2). Both types of elements can form hairpin structures with a length of about 30 bases. Although the actual hairpins have not been demonstrated directly in the native mRNAs, thermodynamic considerations suggest that they exist. In each case, the regions flanking the central consensus motif SCAGUGN-3 are able to form a stem, despite differences in their actual nucleotide sequences (Table 2). In addition, we have shown that various short in vitro transcripts harboring single palindromes, and lacking the possible constraints of folding imposed by flanking sequences, are good targets for IRF binding (Figures 2 and 3). An exception was the first palindrome (Table 2, sequence a), which differs in its central motif and interacts only weakly with IRF. According to the consensus, both

NNN

uu CAGUGN

GG

(9) (h) (0 (k) (1)

NNN

NNNNNNNN

consensus

sequence

cc

Sequences in (a) to (e) correspond to base numbers (a) 3429-3461, (b) 3479-3511, (c) 3662-3914, (d) 3947-3979, and (e) 3994-4026 from the 3’ UTR of hTR mRNA (Schneider et al., 1964). The ferritin sequences are from mRNA for rat light chain (f) (bases 29-61; Leibold and Munro, 1967); rat heavy chain (g) (bases 26-60; Murray et al.. 1967); human light chain (n) (bases 131-163; Santoro et al., 1966); human heavy chain (i) (bases 29-61; Costanzo et al., 1966); bullfrog heavy chain (k) (bases 23-55; Didsbury et al., 1966); and chicken heavy chain (I) (bases 31-63; Stevens et al., 1967). The complementary bases of the palindromes able to form a double strand are indicated on top by arows.

Cell 350

structure and sequence contribute to the specificity of IRF binding. In addition, the central motif and an unpaired cytosine (Casey et al., 1988) appear to be invariable among all sequences listed (Table 2). From the viewpoint of evolution, it is interesting to note that each of the ferritin palindromes is more similar to the group of ferritin elements from other species than it is to the hTR palindromes within the same species. This suggests that an ancestral element may have duplicated prior to divergence of mammals and birds. Accordingly, the 3’ UTR of chicken transferrin receptor mRNA has five conserved palindromes that fit perfectly into this scheme (L.-N. L. Chan, personal communication). We wondered whether IRF is involved in the regulation of mRNA species other than MR and ferritin. We searched the European Molecular Biology Laboratory data bank for elements that conform to the consensus sequence and flanking structure of the IRF binding sites. The survey was made by keeping constant the central consensus and an unpaired @dine, and by permutating all 5 adjacent bp (Table 2). By this analysis, all palindromes listed above were found. The data bank did not contain any additional mRNA sequences conforming to the constraints, This does not preclude a possible involvement of IRF in other regulatory pathways of iron metabolism. If IRF binding sites were frequent in the total cellular mRNA population, one might expect that poly(A)+ RNA would be an efficient competitor in our binding assay. However, weobserved that as much as 2 kg of total mRNA was not sufficient to compete the binding of IRF to 0.1 ng of a labeled MR specific probe (E. W. Mtillner, unpublished data). Taking into account the average sizes of mRNAs and of our probe, we estimated that the abundance of IRF binding sites is less than 0.1% in poly(A)+ mRNA. Biochemical characterization indicates that the cytoplasmic factor interacting with MR and ferritin RNAs has the same binding specificity in vitro. The cross-competition experiments (Figure 3) and the equal molecular weight of the proteins cross-linked by UV to either MR- or ferritin-specific regulatory sequences (Figure 4) suggest that the factors are identical. Moreover, the functional equivalence of ferritin and MR elements has been shown by constructs carrying a MR palindrome in the 5’ UTR of the human growth hormone mRNA (Casey et al., 1988). Currently, we have no formal proof to exclude the possibility that IRF comprises several related proteins. Mouse fibroblast IRF resolves in a doublet of 83 kd and 89 kd in SDS-polyacrylamide gels when cross-linked to either MR- or human ferritin-specific RNA (Figure 4). These sizes are close to the 87 kd protein from rat liver observed to bind the palindrome in the 5’ UTR of rat ferritin mRNAs (Leibold and Munro, 1988). The heterogeneous appearance of RNA-protein complexes formed by mouse cell extracts as resolved in nondenaturing gels suggests a diversity in IRF molecules (Figure 3; Leibold and Munro, 1988). Although under the same conditions a human extract yields only a single complex (Figure 3; Rouault et al., 1988), it cannot be concluded that IRF is less heterogeneous in man. Both human and mouse IRF proteins are

resolved into two bands with molecular weights of 83 kd and 89 kd in SDS-polyacrylamide gels upon UV crosslinking. In gel filtration, IRF activity eluted as a single peak with an approximate molecular weight of 105 kd, indicating that unbound IRF is a monomer. The apparent difference of about 20 kd between the molecular weights of IRF determined in denaturing and nondenaturing conditions can lead to speculations about the presence of a dissociable cofactor. Several RNA binding proteins contain a small RNA, as observed for spliceosomes (reviewed by Maniatis and Reed, 1987), for a component of the complex involved in RNA 3’ end formation (Christofori and Keller, 1988) as well as for poly(A)- and CAP-binding proteins (reviewed by Richter, 1988). Additional work is required to determine the structural components of IRF. It will be of particular interest to find out whether iron inhibits IRF synthesis or whether it represses the activity of a preexisting IRF pool through effects on a cofactor or a posttranslational modification. Preliminary experiments indicate that neither iron nor desferrioxamine are sufficient to modulate IRF activity in vitro. Despite the clear homology between the regulatory elements of MR and ferritin mRNA, their function is different in the context of their native surrounding. The ferritin palindromes located at the 5’ end of the mRNA mediate an inhibition of translation by binding IRF, whereas MR palindromes in the 3’ UTR confer mRNA stability upon binding the cytoplasmic factor. Thus, it appears that IRF is responsible for the coordination of two distinct regulatory events that both play a role in intracellular iron metabolism (Figure 7). High iron levels increase production of ferritin and decrease MR expression. As an overall effect, more iron is deposited into ferritin and less iron can be taken up by the cells through the binding of iron-loaded transferrin to its cell surface receptor. In contrast, at low iron levels, IRF activity is induced, enhancing its binding both to MR and ferritin mRNA. This inverses the balance in favor of an increased iron supply from the cellular deposits and serum. By influencing the expression of two important proteins in iron metabolism, IRF plays a central role in the maintenance of intracellular iron homeostasis. It is essential to realize, however, that iron itself is the main actor in this system. Thus, in activating IRF, iron exerts a feedback regulation on its own level in the cell. Further biochemical characterization of IRF should lead to the elucidation of how specific tissues with an increased demand for iron meet their nutritional requirement for this essential element. In addition, we hope that the present findings will help to define general models for RNA-protein interactions in posttranscriptional regulatory mechanisms. Experlmental Cell Cultura

Procedures and Preparation

of Cytoplwmlc

Extracts

Mouse L cells and transfectants were grown in alpha minimal essential medium supplemented with 10% fetal calf serum. For induction of MR, cells were incubated for 30 hr with medium containing 50 pM desferrioxamine (Desferal, gift from Ciba-Geigy, Easel, Switzerland). Where appropriate, ferric ammonium citrate was added to the cultures for 30 hr at 10 pglml. Under these conditions, cell-plating efficiency and cell

Regulation 381

of Transferrin

Receptor

mRNA

Stability

cycle distribution were not affected. Actinomycin D and cycloheximide were at concentrations of 5 pM and 40 PM. respectively. The effectiveness of transcriptionand protein-synthesis inhibition was checked by pulse labeling with 3H-uridine and FSjmethionine, respectively. Radiolabeled DNA and protein were precipitated by trichloroacetic acid, collected on glass fiber filters, and quantitated by scintillation counting. For preparatim of cytoplasmic extracts, 2.5 x IO’ L cells were lysed at 4% in 200 PI of extraction buffer that contained 10 mM HEPES (pH 7.5), 3 mM MgCl*, 40 mM KCI, 5% glycerol, and 1 mM dithiothreitol supplemented with 0.2% NP40. After lysis, samples were diluted g-fold with extraction buffer, thus giving a final protein concentration of approximately 0.5 mg/ml. Nuclei were removed by centrifugation at 10,000 x g for 1 min and the supernatant stored at -8oOC. Cytoplasmic extract from human placenta was prepared as described by Booth and Wilson (1981) and diluted to a final protein concentration of 0.5 mglml with extraction buffer. t

Plasmid

Constructions

Fragments from deletion mutants of the full-length hTR cDNA clone pcD-TRl (Ki.ihn et al., 1984; Mtillner and Kiihn, 1968) were subcloned into pSPT18/19 (Boehringer Mannheim) in the + orientation relative to the promoter for T7 RNA polymerase. The resulting plasmids cuntain the following hTR 3’ UTR sequences, numbered according to Schneider et al. (1984): pSPTqR11 2988-BamHI site 29 bases 3’to the poly(AT) region of the pcD-vector (Okayama and Berg, 1983); pSPTTR12 2986-3499, 3578-BamHI; pSPT-TR21 3421-3597, 3848-4048; pSPT-TR22 3511-3876; pSPT-TR23 3421-3876; pSPT-TR24 35114048; pSPT-TR25 3393-3884; pSPTTR28 3393-3530; pSPT-TR27 351 l-3596; pSPT-IR28 3525-3567; pSPT-TR29 3822-4048; pSPTTR31 3881-3970; pSPTTR32 3822-3928, 3979-4048; pSPTTR33 3881-3928, 39794048; and pSPT-TR34 3979-4048. pSPT-TR13 has the same insert as pSPT-TRll but in the - orientation. pSPT-act has the 1.1 kb Pstl-Pstl fragment of the mouse B-actin cDNA in the + orientation (Minty et al., 1983). pSPT-fer contains an oligonucleotide corresponding to bases 31-58 of the S’untranslated region of human ferritin heavy chain mRNA (Costanzo et al., 1988). Oligonucleotides for pSpTTR28 and pSPT-fer were synthesized on a DNA synthesizer (model 3808, Applied Biosystems).

Preparation

Polysome

and

Gmdlents

Cytoplasmic extracts were prepared from 5 x 10’ mouse L cells transfected with the regulated MR cDNA clone pTR-81 (Miillner and Kiihn. 1988) in 1 ml of extraction buffer (see above) supplemented with 1000 U/ml of RNasin, 150 pgIml of cycloheximide, 1 mM pheny-lmethylsulfonyl fluoride, 20 mM dithiothreitol, and 5 mg/ml of heparin. Mitechondria and nuclei were removed by centrifugation at 10,000 x g for 10 min. Separation of polysomes from the postmitochondrial supernatant was carried out on preformed 12 ml, linear 15%40% sucrose gradients (Luthe, 1983) in extraction buffer containing 0.5 mg/ml of heparin, 150 pg/ml of cycloheximide. and 20 mM dithiothreitol. The samples were centrifuged at 4OC for 2 hr at 38,000 rpm in a Kontmn TST 41.14 rotor. Immediately after centrifugation, gradients were separated into 18 fractions on an ISCO fractionator (Lincoln, NB). After phenol-chloroform extraction, the integrity of RNA in each fraction was assessed by ethidium-bromide staining of RNA separated in a nondenaturing 1% agamse gel. Subsequently, the samples were transferred to GeneScreen plus nylon filters (DuPontlNEN, Boston) and immobilized by UV fixation. The filters were hybridized with either the central 3 kb Hindlll-Hindlll fragment of pcD-TRl (Kiihn et al., 1984) or, as a control, with the 0.3 kb Hhal-EcuRI fragment (positions 23-317) of mouse 62microglobulin cDNA clone p82-m2 (Daniel et al., 1983).

Acknowledgments We thank Christine Meyer for technical assistance, and Drs. Victor Jongeneel and Otto Hagenbuchle for critical reading of the manuscript. We acknowledge the access to the computer programs at the Friedrich Miescher Institute, Base& Switzerland. This research was supported by the Fores Foundation, the Swiss League for Cancer Research, grant FOR.336.86.1, and by the Swiss National Science Foundation, grant 3.635-0.87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adve&ement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

of RNA Transcripts

Transcription reactions were performed with 1 pg of linearized plasmid in the presence of 100 pCi of [u-~~PJCTP (Amersham, 800 CilmM) and 2.5 mM unlabeled ribotriphosphates with 20 U of T7 RNA polymerase (Boehringer Mannheim) in a final volume of 10 ~1. Samples were incubated for 1 hr at 40°C. Specific activity of transcripts performed in this way is 1.3 x IO9 dpmlpg. For unlabeled RNAs, all triphosphates were at a concentration of 2.5 mM. Full-length transcripts were purified by agarose gel electrophoresis or chromatography on Sephacryl S-200 (Pharmacia, Uppsala, Sweden).

Analysis

with 0.5 mg of transferrin (M, = 80,000). catalase (M, = 232,000), thyroglobulin (M, = 669poO) as protein standards.

of RNA-Protein

Interactions

Binding reactions were carried out as described by Leibold and Munro (1988). Fifty micrograms of cytoplasmic extract and 0.15 fmol of labeled RNA transcript (1.3 x 106 dpmlng) were incubated in a final volume of 20 ~1 of extraction buffer for 30 min at room temperature. Subsequently, unprotected RNA was digested for 10 min by 1 U of RNAase T, (1 U/II). Heparin (50 mg/ml) was added to a final concentration of 5 mglml for another 10 min. RNA-protein complexes were resolved in 4% nondenaturing polyacrylamide gels run at 10 Vlcm for 2 hr in the cold as described (Konarska and Sharp, 1986). Gels were dried and autoradiographed at -8oOC. For UV cross-linking of RNA and protein, the reaction mixtures were put on ice immediately after the addition of heparin and irradiated 5 cm below a UV lamp for 30 min (Philips TUV 15 W, 10 mW/cm2). The samples were analyzed by electrophoresis on a 10% SDS-polyacrylamide gel under reducing conditions. For gel filtration, 200 PI samples with RNA-protein complexes formed from 50 pg of protein and 0.5 ng of =P-labeled RNA were separated on a Superose 6 column by FPLC (Pharmacia, Uppsala, Sweden) in extraction buffer. To eliminate nonspecific binding of proteins to RNA, samples were loaded in the presence of 200 rg of E. coli rRNA and 5 mglml of heparin. The apparent M, of RNA-protein complexes and the unbound factor was determined by calibrating every run

Received

April 13, 1989.

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