(Received for publication, February 21, 1997, and in revised form, May 29, 1997)
From the Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany
In cultured rat hepatocytes the degradation of
phosphoenolpyruvate carboxykinase mRNA might be regulated by
protein(s), which by binding to the mRNA alter its stability. The
3-untranslated region of phosphoenolpyruvate carboxykinase mRNA as
a potential target was used to select RNA-binding protein(s) from rat
liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA
3
-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the
24.5-kDa protein revealed its identity with rat ferritin light chain.
Binding of ferritin to RNA was abolished after phosphorylation with
cAMP-dependent protein kinase and was augmented after
dephosphorylation with alkaline phosphatase. Weak binding was observed
in extracts from okadaic acid-treated cultured hepatocytes compared
with untreated cells. Preincubation of ferritin with an
anti-phosphoserine or an anti-phosphothreonine antibody attenuated
binding to RNA, while an anti-phosphotyrosine antibody generated a
supershift indicating that phosphoserine and phosphothreonine but not
phosphotyrosine residues were in close proximity to the RNA-binding
region. Ferritin is the iron storage protein in the liver. Binding of
ferritin to RNA was diminished in the presence of increasing iron
concentrations, whereas the iron chelator desferal was without effect.
It is concluded that ferritin might function as RNA-binding protein and
that it may have important functions in the general regulation of
cellular RNA metabolism.
In the regulation of RNA metabolism protein-RNA interactions play
a central role. This is well documented for the splicing process, which
eliminates intervening sequences from the primary transcript in the
ribonucleoprotein particle of the spliceosome (1), for the transport of
RNA from the nucleus to the cytoplasm (2), for translational
initiation, which is mediated by protein factors recognizing the
initiation region on the mRNA and positioning the ribosome to the
translational start site (3), for the storage of translationally
inactive mRNA (4) or for spatial localization of mRNA in the
cell (5). Even if the mechanism and regulation of mRNA degradation
are far from being understood, it is clear that RNA-protein
interactions have a key role (6, 7). Besides its function in RNA
transport and translation the poly(A) tail has been shown to protect
poly(A) carrying mRNAs from rapid degradation by interaction with
the poly(A)-binding protein PABP (8, 9). Many mammalian mRNAs
contain AU-rich portions in their 3-untranslated region, which after
being occupied by AU-binding proteins render the RNA instable
(10-12).
In cultured rat hepatocytes and in rat hepatoma cells the expression of
the key control enzyme in gluconeogenesis, phosphoenolpyruvate carboxykinase (PCK),1 was
stimulated at the transcriptional level by glucagon (13) or cAMP (14,
15). Insulin, the glucagon antagonist at large, inhibited the glucagon-
(cAMP-)stimulated gene transcription (13, 16). Yet, besides
transcriptional regulation the expression of PCK is also regulated at
the level of mRNA degradation. In cultured rat hepatocytes insulin
and the proinflammatory cytokine interleukin 6 accelerated the
degradation of glucagon-induced PCK mRNA (13, 17). The acceleration
of PCK mRNA degradation was probably due at least in part to the
insulin-enhanced expression of a
ribonuclease.2 In FTO-2B rat
hepatoma cells cyclic AMP stabilized PCK mRNA (18, 19). The
stability of PCK mRNA seems to be regulated by proteins binding to
the 3-untranslated region of the mRNA. In cultured rat hepatocytes
binding of a protein was enhanced after glucagon treatment, which
correlated with PCK mRNA accumulation. Insulin prevented the
increase in protein binding, which correlated with the decrease in PCK
mRNA levels (20, 21). In FTO-2B cells cAMP caused reduced binding
of a protein to the 3
-UTR of PCK mRNA (22).
It was the goal of this investigation to identify and characterize protein(s), which might contribute to the regulation of PCK mRNA degradation. By gel retardation assays a cytosolic protein binding to PCK mRNA was identified, which was purified and biochemically characterized. Peptide sequence analysis showed identity of the protein with rat ferritin light chain. The binding of ferritin to RNA was regulated by phosphorylation/dephosphorylation and by iron. Competition experiments showed that ferritin did not bind preferentially to PCK mRNA but to a variety of different RNAs. It is suggested that ferritin may play a regulatory role in PCK mRNA degradation as well as in the general cytosolic mRNA turnover. The identification of ferritin as RNA-binding protein is discussed in view of an old hypothesis and recent publications, which proposed ferritin to play a role in the general metabolism of cytosolic RNA (23, 24).
Liver cells were isolated from male fed Wistar rats (200-250 g) by the collagenase perfusion technique. Cells were seeded on 6-cm diameter plastic culture dishes (Greiner, Nürtingen) and maintained in culture in the presence of 100 nM dexamethasone (Sigma, Deisenhofen) and 0.5 nM insulin (Sigma, Deisenhofen) to improve cell viability as described (13). After 48 h of culture, cells were washed twice with medium containing only 100 nM dexamethasone. Fresh medium containing in addition 0.1 nM glucagon (Serva, Heidelberg) was added and the culture was continued for another 2 h. Then insulin at 10 nM and/or the phosphatase inhibitor okadaic acid (ICN, Meckenheim) at 0.5 µM and/or the transcriptional inhibitor actinomycin D at 10 µg/ml (Sigma, Deisenhofen) were added for another 3 h. Finally the cells were harvested for preparation of protein extracts.
Preparation of Cytosolic Extracts from Cultured Rat HepatocytesCells from one culture dish were washed twice with
ice-cold phosphate-buffered saline, scraped off in 800 µl of
homogenization buffer containing 10 mM Tris/HCl, pH 7.4, 250 mM sucrose, 0.5 mM CaCl2, 0.5 mM dithioerythreitol (Serva, Heidelberg), 0.5 mM phenylmethylsulfonyl fluoride (Serva, Heidelberg), and
homogenized in a glass homogenizer with a motor-driven Teflon pestle
for 6 min at maximal speed. The homogenate was centrifuged at
1,500 × g for 20 min, the resulting supernatant at
8,500 × g for 15 min, and the then resulting
supernatant at 120,000 × g for 1 h to obtain a
post-polysomal, cytosolic extract (S120 extract). After heating to
50 °C for 15 min the crude extract was centrifuged at 14,000 × g for 15 min (Fig. 3). The resulting supernatant was brought
to 35% saturation with ammonium sulfate and stirred on ice for 30 min.
After centrifugation of the slurry for 20 min at 14,000 × g the supernatant was brought to 65% saturation with ammonium sulfate and stirred again on ice for 15 min. The slurry was
centrifuged at 14,000 × g for 20 min, the pellet was
suspended in dialysis buffer, which contained 10 mM
Tris/HCl, pH 7.4, 10% glycerol, 0.5 mM each of
dithioerythreitol and phenylmethylsulfonyl fluoride and the extract was
dialyzed (cut off Mr 3,500, Spectrum Medical
Industries) overnight at 4 °C (cytosolic extract CE) (Fig. 3).
Protein concentration was determined by a commercial kit (Bio-Rad, München). The extract was stored at 70 °C. The same
procedure was performed for the preparation of cytosolic extracts from
whole rat liver or other organs as indicated. In this case tissue was homogenized in 10 volumes of homogenization buffer.
Purification of a RNA-binding Protein from Rat Liver
3.5 g of swollen DEAE Sephadex A-25 (Pharmacia LKB Biotechnologies Inc., Freiburg) pre-equilibrated with buffer A (10 mM Tris/HCl, pH 7.4, and 0.5 mM EDTA) was incubated in a batch procedure for 30 min at 4 °C with 10 ml of cytosolic extract (CE) containing about 100 mg of total protein. Buffer A containing unbound proteins was collected and combined with buffer A resulting from two subsequent washes of the gel. Bound protein was eluted by washing with buffer B (10 mM Tris/HCl, pH 7.4, 0.5 mM EDTA, and 1 M NaCl). Eluted protein and the unbound material were concentrated separately by ultrafiltration (Centriplus 100, Amicon Inc., Beverly, MA). The unbound protein fraction called FT1 (for historical reasons because this procedure was processed in a column at the beginning of the study) was further processed with fast protein liquid chromatography on the anion exchange matrix Source 30 Q (Pharmacia, Freiburg) in an HR 5/5 column (Pharmacia, Freiburg) (Fig. 3). The column was equilibrated with three successive washes with buffer A. The sample was loaded onto the column and the resin washed with buffer A until protein was no longer detectable in the flow-through (FT2). Bound protein was eluted in a linear gradient ranging from 0 to 1 M NaCl at a flow rate of 0.5 ml/min. Fractions (1 ml) were assayed for RNA binding activity in the gel retardation assay as described below. RNA binding activity containing fractions were pooled (range of fractions 6-10) (Source extract SO), concentrated, and equilibrated with buffer A by ultrafiltration on Centricon 100 and loaded onto a Superdex 200-pg column (Column volume, Vc = 135 ml) equilibrated with buffer A (Fig. 3). Elution was performed in buffer A at a flow rate of 0.25 ml/min and 0.5- or 0.6-ml fractions were collected and assayed for RNA binding activity (range of fractions 36-38) (Superdex extract). For molecular mass determination the Superdex column was calibrated with standard proteins in a gel filtration calibration kit (Sigma, Deisenhofen). SDS-polyacrylamide electrophoresis under reducing conditions revealed that the Superdex extract contained one prominent protein band and only minor contaminations. Therefore it was used to raise an antiserum against the purified RNA-binding protein in rabbits.
Preparative Isoelectric Focusing200-250 mg of protein in the S120 extracts was incubated in a final volume of 50 ml in focusing puffer (10 mM Tris/HCl, pH 7.4, 2 M urea, 2% Ampholyte pH 3-10) and filled into the precooled (4 °C) Rotofor® cell (Bio-Rad, München). Isoelectric focusing was performed at constant power of 12 watts for 5-6 h. During this time voltage increased from 290 to 900 V and the current decreased from 41 to 13 mA. At the end of the run samples were fractionated as described in the manufacturer's manual and pH was measured in each sample. To separate ampholytes from protein, the samples were incubated in 1 M NaCl for 30 min at 4 °C. Then they were dialyzed against 10 mM Tris/HCl, pH 7.4, 0.5 mM EDTA, and 10% glycerol overnight. Each fraction was assayed for RNA binding activity as described below. Five different focusing buffers were tested containing CHAPS, urea, glycerol, digitonin, and/or Tris/HCl at different concentrations and combinations to minimize denaturation of protein during the focusing procedure. The Tris/HCl buffer described here yielded the best results.
Preparation by PCR of Double-stranded DNA Templates for in Vitro TranscriptionPlasmid pBS-PCK722 containing a 722-base pair
PstI-EcoRI PCK cDNA fragment, which
represented 120 translated and 602 untranslated bases of the 3-portion
of PCK mRNA (bases 1890-2611, Ref. 14), was used as the template
for the production of PCR products. To obtain various DNA templates for
the generation of PCK transcripts of different lengths and regions from
the PCK mRNA 3
-UTR the following primers were used for PCR: sense
primer 1 and 2 contained at the 5
-end the T7-RNA polymerase promotor
sequence (5
-TAATACGACTCACTATAG-3
) followed by genuine PCK mRNA
sequences from bases 2010 to 2030 and 2265 to 2291 (14). Antisense
primers 1 and 2 contained genuine PCK mRNA sequences from bases
2235 to 2259 and 2583 to 2603 (14). The combinatorial use of the primer
pairs sense primer 1 and antisense primer 2, sense primer 1 and
antisense primer 1, sense primer 2 and antisense primer 2 yielded PCR
products PCK1, PCK2, and PCK3, which contained either the total 3
-UTR
(bases 2010-2603), the 5
-portion (bases 2010-2259), or the
3
-portion (bases 2265-2603) of the PCK mRNA 3
-UTR (14). PCR was
performed with 1 ng of pBS-PCK722 and 80 pmol of each primer in 10 mM Tris/HCl buffer, pH 8.3, containing 50 mM
KCl, 1.5 mM MgCl2, 1 mM of each
dCTP, dTTP, dATP, and dGTP and finally 1 unit of Taq DNA
polymerase. PCR was performed in 35 cycles (45 s at 95 °C, 45 s
at 55 °C, 60 s at 72 °C). The resulting PCR products were
taken as templates for a second round of PCR under the same conditions.
These PCR products were purified by a commercial kit (Qiagen,
Düsseldorf) and the DNA content was measured
spectrophotometrically.
The PCR products PCK1, PCK2, and
PCK3 were used as templates to generate PCK1, PCK2, and PCK3
transcripts. Transcription reactions were carried out with 10 nM PCR products (see above) in a final volume of 40 µl in
transcription buffer (Stratagene, Heidelberg), containing 0.5 mM adenosine-, cytidine-, and guanosine 5-triphosphate each, 0.025 mM uridine 5
-triphosphate, 55 pmol of uridine
5
-[
-32P]triphosphate (600 Ci/mmol) (ICN, Meckenheim),
10 mM dithioerythreitol, and 25 units of human placental
ribonuclease inhibitor (Amersham, Braunschweig). The reaction was
started by addition of 25 units of T7 RNA polymerase (Stratagene,
Heidelberg). After incubation for 45 min at 37 °C another 25 units
of T7 RNA polymerase were added and the reaction was continued for 45 min at 37 °C. The reaction was terminated by adding 10 µg of tRNA
(Boehringer, Mannheim) and 25 units of human placental ribonuclease
inhibitor. Subsequently template DNA (PCK1, 2, or 3) was digested with
1 unit of RNase-free DNase I (Boehringer) for 15 min at 37 °C. The
mixture was brought to 300 mM sodium acetate and 8 mM dithioerythreitol; the volume was adjusted to 70 µl
with diethyl pyrocarbonate-treated water. Transcripts were purified by
extraction of proteins with 70 µl of buffered phenol (Amresco,
Solon), pH 4.3. Phases were separated by centrifugation for 5 min in a
microcentrifuge and the aqueous phase was applied to a Nick column
(Pharmacia, Freiburg) to remove unincorporated nucleotides. Human
histone H1° and mouse arylsulfatase A transcripts were generated by
the same procedure using plasmid DNA kindly provided by Drs. D. Doenecke (Göttingen) and V. Gieselmann (Kiel).
Standard binding reaction was carried
out in a final volume of 40 µl with 40 µg or as indicated total
protein and about 5 ng of 32P-radiolabeled RNA in binding
buffer containing 10 mM Tris-HCl, pH 7.4, 100 mM KCl, 3 mM MgCl2, 3 mM EDTA, and 40 µg of RNase A (Boehringer, Mannheim).
Samples were incubated for 90 min at 37 °C. After addition of 5 µl
of electrophoresis buffer containing 10% glycerol and 0.01%
bromphenol blue, reaction mixtures were separated on a 5% native
polyacrylamide gel (34 × 16 cm) for 2-3 h at 20 mA (about 6 volt/cm) at room temperature. After electrophoresis gels were dried and
exposed to Hyperfilm MP (Amersham, Braunschweig) in general overnight
at 70 °C or analyzed with a PhosphorImager (Molecular Dynamics,
Krefeld). Competition experiments were carried out by addition of
unlabeled competitor RNAs to the binding reaction 3 min prior to the
addition of the labeled transcript.
In the phosphorylation reaction 40 µg of protein in the CE was treated with 80 units of the catalytic subunit of the cAMP-dependent protein kinase (Sigma) for 30 min at 37 °C in 40 µl of phosphorylation buffer containing 10 mM Tris/HCl, pH 7.4, 4.8 mM MgCl2, and 0.85 mM ATP. In the dephosphorylation reaction 40 µg of protein in the CE was treated with 7 units of calf intestinal alkaline phosphatase (U. S. Biochemical Corp., Cleveland, OH) for 30 min at 37 °C in 40 µl of dephosphorylation buffer containing 10 mM Tris/HCl, pH 7.4, and 0.01 mM ZnCl2. Controls were performed without addition of kinase or phosphatase.
Detection of Phosphorylated Amino Acids by Anti-phosphoamino Acid Antibodies1 µg of protein in the Superdex extract (Fig. 4) was
incubated with 0.5 or 1 µg of anti-phosphoserine,
anti-phosphothreonine (Biomol, Hamburg), or anti-phosphotyrosine
antibody (Amersham, Braunschweig) or with an unrelated antibody in 10 mM Tris/HCl, pH 7.4, and 0.05% Triton X-100 in a final
volume of 10 µl at 4 °C overnight. After the incubation the
mixture was submitted to the standard RNA binding assay.
Immunoprecipitation of Ferritin with an Anti-ferritin Antibody
40 mg of protein A coupled to Sepharose CL-4B beads (Pharmacia, Freiburg) was swollen in 1.5 ml of TS buffer (15 mM Tris/HCl, pH 7.4, 150 mM NaCl) for 4 h at room temperature. The swollen gel was collected by centrifugation and washed 3 times in TS. The final pellet (200 µl) was resuspended in 200 µl of TS and combined with 20 µl (90 µg) of anti-ferritin antiserum. The mixture was incubated overnight at 4 °C and subsequently for 4 h at room temperature. 60 µl from this mixture were combined with 40 µg of protein in the CE and incubated overnight at 4 °C. Precipitates were pelleted by centrifugation at 14,000 × g. The supernatant was taken off (supernatant 1) and the pellet was washed 2 times with 30 µl of TS. The resulting supernatants were combined with supernatant 1. 25 µg of protein in this ferritin-free CE was applied to the gel retardation assay.
MiscellaneousSDS-polyacrylamide gel electrophoresis was performed according to Laemmli (25). Mass spectroscopy and sequencing of tryptic peptides of the purified protein were conducted by Drs. B. Schmidt and T. Selmer (Göttingen, Germany). Antiserum against the purified protein from the Superdex extract was generated in rabbits by Eurogentec (Seraing, Belgium).
To identify and characterize proteins, which might be
involved in the regulation of PCK mRNA degradation, a gel
retardation assay was established using cytosolic protein extracts from
rat liver and a 32P-labeled transcript, which was produced
by in vitro transcription from a PCR-generated DNA fragment
containing the entire 3-untranslated region of PCK mRNA (PCK1
transcript, 594 bases long; position 2010 to 2603 in PCK mRNA; Ref.
14).
When the binding reaction was performed in the absence of RNase A two
dominant RNA-protein complexes (complex 1 and 2) formed with PCK1
transcript and cytosolic protein(s) from rat liver. While formation of
complex 2 did not require the presence of Mg2+ complex 1 formed only in the presence of both Mg2+ and EDTA (Fig.
1A, lanes 3-5 versus lanes
4-6). Addition of RNase A yielded the specific formation of only
complex 1 alone, essentially only in the presence of equimolar
concentrations of a divalent cation (Mg2+ or
Mn2+ or Ca2+ or Zn2+) and EDTA as
shown representatively for Mg2+/EDTA (Fig. 1A, lane
10 versus lanes 7-9). This complex was only obtained with protein
from rat liver cytosol and not with bovine serum albumin or with
nuclear protein extracts (Fig. 1A, lanes 11 and
12).
Complex formation required incubation at 37 °C for at least 15 min (Fig. 1B, lanes 4-6) and did not occur at 4 °C (Fig. 1B, lanes 1-3), indicating that a temperature-sensitive step was involved in RNA-protein complex formation. Heating the cytosolic extract to 70 °C abolished complex formation (not shown). Pretreatment of the extracts with proteinase K did not destroy the RNA binding activity of the protein (Fig. 1B, lane 11). Pretreatment of the extracts with a protease mixture containing proteinase K, trypsin, and Pronase E prevented complex formation (not shown). These data show that complex 1 was due to an interaction of protein(s) with PCK1 transcript.
RNA-protein interactions can be expected to be dependent on salt concentration and pH. Binding of the rat liver cytosolic protein to PCK1 transcript was still observed at 0.4 M KCl but was lower compared with 0.1 M KCl (Fig. 1B, lane 6 versus 7). Protein binding to PCK1 transcript was stronger at pH 4.0 compared with pH 9.0 (Fig. 1B, lanes 8-10). Preparative isoelectric focusing (IEF, "Experimental Procedures") enriched the RNA binding activity in fractions between pH 4.0 and pH 5.2, which showed that the RNA-binding protein was acidic.
The binding activity containing IEF fractions were used for an estimate of the binding affinity of the protein to RNA. Increasing the amount of protein from 10 to 100 µg at a constant amount of the PCK1 transcript (16 fmol) increased complex formation in a sigmoidal manner. Half-maximal protein binding was reached at 38 µg, maximal binding at 80 µg of protein (not shown). The binding protein from rat liver (which was identified as rat ferritin light chain, see below) was enriched by 15-fold by the IEF procedure (5-fold in the protein extract over the crude liver homogenate and 3-fold in the IEF fraction over the protein extract). Since 1 g of liver contains 180 mg of protein (18% of the wet weight) (26) and 600 µg of ferritin = 1.5 nmol (molecular mass 400 kDa) (27), 1 mg of liver protein would provide 8.33 pmol of ferritin, and 1 mg of IEF fraction protein, being enriched in binding activity by 15-fold, would provide 125 pmol of ferritin. Thus, in 40 µl of buffer half-saturation of the PCK1 transcript (16 fmol/40 µl = 0.4 nM) was reached by ferritin at 120 nM (38 µg of IEF protein = 4.75 pmol of ferritin/40 µl).
Specificity of Cytosolic Protein Binding to RNABinding of
cytosolic protein to PCK1 transcript should be hindered in the gel
retardation assay by RNA molecules, which share sequence and/or
structure similarities with PCK1 transcript. To study the binding
preference of the protein for different RNAs, the gel retardation assay
with PCK1 transcript was performed in the presence of single-stranded
RNA homopolyribonucleotides (poly(A), poly(C), poly(G), and poly(U))
(Fig. 2A, upper panel). In the presence of poly(U) (500 and 1000-fold excess by weight) binding of
protein to PCK1 transcript was abolished and with tRNA (2000 and
4000-fold molar excess) binding was effectively attenuated. Poly(A)
competed for protein binding to a lesser extent than poly(U), whereas
poly(C) exhibited weak and poly(G) no competition for binding. Binding
to labeled PCK1 transcript was also investigated in the presence of
unlabeled transcripts of PCK1, PCK2, and PCK3, which contained either
the entire 3-UTR of PCK mRNA (PCK1 bases 2010-2603) or the 5
(PCK2 bases 2010-2259) or the 3
(PCK3 bases 2265-2603) (14) part of
it in a 150-1200-fold molar excess (Fig. 2, bottom part).
With all three unlabeled transcripts binding of cytosolic protein to
PCK1 transcript was effectively attenuated. However, when the PCR DNA
fragments, which were used as templates for the generation of the
transcripts, were included in a 500-fold excess by weight in the
binding reaction, no interference with protein binding was
observed.
Protein binding to PCK1 transcript was compared with binding to the
3-UTR of the human histone 1° (HIS) mRNA, which encodes a
nuclear protein, and a portion of the 3
-UTR of the mouse arylsulfatase A mRNA, which encodes a lysosomal protein. With cytosolic protein from rat liver HIS and arylsulfatase A transcripts formed a complex, which had the same electrophoretic mobility as the complex with PCK1
transcript (Fig. 2B).
The tissue distribution of the cytosolic RNA-binding protein was studied. Cytosolic protein extracts from rat liver (LI), kidney (KI), spleen (SP), and lung (LU) were applied to the gel retardation assay with the PCK1 transcript. Whereas rat liver extracts exhibited strong protein binding to the PCK1 transcript, extracts from kidney, spleen, and lung showed weak but significant binding forming a complex with slightly higher mobility in the case of spleen and lung (Fig. 2C).
These data show that the cytosolic RNA-binding protein was prevalent in
rat liver and that the protein bound specifically to all transcripts
investigated but not to DNA. The fact that poly(U) and to a lesser
extent poly(A) competed for protein binding might indicate that
single-stranded A- and/or U-rich sequences were involved in the contact
of the protein to RNA, which are present in multiple regions in the
3-untranslated part of PCK mRNA (14).
The RNA-binding protein was purified by sequential
chromatography of cytosolic protein of rat liver on the weak anion
exchange resin DEAE 25, the strong anion exchange material Source Q,
and the final gel filtration over Superdex 200-pg column (Fig.
3). The enrichment of the RNA-binding
protein from the CE was assessed in the gel retardation assay with PCK1
transcripts by counting the radioactivity in the RNA-protein complex
using a PhosphorImager. Whereas after the second anion exchange
chromatography on Source Q the RNA binding activity was enriched in the
range of fractions 7-15 about 10-fold over the cytosolic protein
extract, it was enriched only about 5-fold after gel filtration on
Superdex 200-pg column in the range of fractions 36-38. The
RNA-binding protein eluted from the Superdex column in a molecular mass
range of about 400 kDa. SDS-polyacrylamide gel electrophoresis analysis
of this material under reducing conditions revealed that this fraction contained one prevalent protein of 24,500 Da. Two further protein bands
of molecular masses of 59,500 Da and of about 150,000 Da were visible
in the SDS-polyacrylamide gel electrophoresis. However, after
incubation for 20 min at 95 °C in -mercaptoethanol these two
bands vanished and only the 24.5-kDa protein appeared (not shown). This
indicated that the RNA-binding protein had a native molecular mass of
about 400 kDa and consisted of identical subunits with a molecular mass
of 24.5 kDa each. Mass spectroscopy of the purified protein revealed a
molecular mass of 21.2 kDa.
The Superdex-purified native protein was processed for sequencing by carboxymethylation under reducing conditions and was subsequently digested with 1% trypsin. After fractionation of the tryptic digest by high performance liquid chromatography a suitable peptide was chosen for sequencing. Sequencing was terminated after completion of 9 amino acids. The resulting peptide sequence ALFQDVQKP was used for a homology search in the Swiss protein sequence data bank. The search revealed sequence identity with amino acids 76-84 in the rat ferritin light chain (Swiss-Prot AC: P02793).
Gel retardation assays were performed with commercial ferritin (Sigma; 99% pure) and PCK1 transcript and compared with gel retardation assays with the protein purified here from rat liver. Both protein preparations formed a protein-RNA complex with PCK1 transcript, which had the same electrophoretic mobility. Complex formation with the commercial ferritin required the same divalent cation, EDTA, and pH conditions as the purified ferritin from rat liver (data not shown). The purified protein was used to generate a polyclonal antibody. In Western blots this antibody detected both the commercial ferritin and the ferritin purified in this study.
The antibody was incubated overnight at 4 °C with the CE or the purified protein preparation (Fig. 4A, lanes 1-4). In gel retardation assays with the preincubated protein preparations and with PCK1 transcript, complex 1 did not form, indicating that the ferritin antibody prevented protein binding to PCK1 transcript. To show that this was not due to an interaction of the antibody with PCK1 transcript, which could unspecifically prohibit complex formation, ferritin was immunoprecipitated with Sepharose CL-4B protein A-coupled antibody from the CE. Depletion of ferritin from CE by immunoprecipitation with protein A abolished complex formation with the PCK1 transcript (Fig. 4B, lanes 1 and 2) demonstrating an interaction of ferritin with PCK1 transcript.
An Additional Factor Besides Ferritin Was Necessary for Efficient RNA-Protein Complex FormationFerritin formed a RNA-protein
complex with PCK1 transcript. This complex was stronger with binding
activity containing fractions from the anion exchange chromatography
compared with the purified Superdex 200-pg fraction, indicating the
loss of a putative factor, which promoted complex formation between
ferritin and the PCK1 transcript (Fig. 3). 15 µg of protein of
cytosolic protein extract were subjected to the gel retardation assay
with PCK1 transcript and increasing amounts of Superdex extract
fractions 36-38 (Fig. 5A).
The combination of 15 µg of protein CE with 4.8 µg of protein Superdex extract stimulated complex formation 10-fold compared with
Superdex extract alone. This implies that the CE contained a component,
which was lost during gel filtration. Therefore the gel retardation
assay using PCK1 transcript and 1 µg of protein Superdex extract was
completed with fractions from the Superdex chromatography following the
Superdex extract fraction 37 (Fig. 5B). Addition of an
aliquot derived from the seventh fraction after the ferritin-containing
fraction (i.e. fraction 44 of total fractions) increased
complex formation 3-fold. This fraction corresponded to a molecular
mass range of about 180,000 Da. It obviously contained a factor capable
of stimulating complex formation between ferritin and PCK1 transcript.
In the absence of ferritin, protein from this fraction alone did not
form any complex with PCK1 transcript. The nature of the factor remains
at present unknown because it had not been further purified and
characterized. It is, however, clear that the factor does not act on
ferritin binding to RNA by increasing the amount of binding protein but
by enhancing its affinity or its activity. This can be concluded
because protein binding of the combined 4.8 µg of Superdex extract
and of 15 µg of CE was not additive to protein binding of each of the
fractions alone (Fig. 5A). Moreover, in the experiment
described in Fig. 5B the amount of protein in Superdex
extract was kept constant (1 µg) and only the addition of fraction 44 increased protein binding.
In Vitro Phosphorylation/Dephosphorylation Regulated Complex Formation between Ferritin and RNA
If ferritin should play a role
in the regulation of mRNA turnover, it is reasonable to assume that
its binding to RNA should be regulated by
phosphorylation/dephosphorylation. To confirm this assumption, the
purified ferritin was treated in vitro in the presence of
ATP with the catalytic subunit of cAMP-dependent protein
kinase prior to the binding reaction. This treatment abolished protein
binding to PCK1 transcript (Fig.
6A, lanes 1 versus 2). Treatment with alkaline phosphatase prior to the binding reaction enhanced binding of the purified ferritin to PCK1 transcript (Fig. 6A, lanes 3 versus 4). The loss of RNA binding activity of
ferritin after treatment with the catalytic subunit of
cAMP-dependent protein kinase could be restored by
subsequent treatment with alkaline phosphatase (not shown). The data
show that the RNA binding activity of ferritin purified from rat liver
was regulated by phosphorylation/ dephosphorylation.
To specify which amino acid residues were phosphorylated, the purified ferritin was incubated overnight with specific antibodies against phosphoserine, phosphothreonine, and phosphotyrosine or with an unrelated antibody. After this incubation the gel retardation assay was performed with the PCK1 transcript. Incubation with the anti-phosphotyrosine antibody generated a "supershift" (Fig. 6B) indicating that the ferritin contained phosphorylated tyrosine residues. Binding of the antibody to phosphotyrosines did not interfere with protein binding to RNA. In contrast, preincubation with the anti-phosphoserine or anti-phosphothreonine antibodies decreased complex formation (Fig. 6B) indicating that binding of the antibodies to phosphorylated serine and threonine residues in the protein interfered with the binding to RNA. Apparently, binding of the antibody to phosphoserine or phosphothreonine masked the RNA-binding region. Unrelated antibodies did not affect protein binding (not shown).
Inhibition of Protein Phosphatases Decreased Binding of Ferritin to RNA in VivoIn cultured rat hepatocytes glucagon increased PCK
mRNA levels transiently to a maximum after 2 h. When insulin
was given at the maximal increase at 2 h it accelerated PCK
mRNA degradation (13). The serine/threonine phosphatase inhibitor
okadaic acid given at the maximal increase at 2 h inhibited the
degradation of PCK mRNA in the absence and presence of insulin,
indicating the involvement of dephosphorylation events in the control
of PCK mRNA degradation.2 Therefore, to demonstrate
that phosphorylation and dephosphorylation may also play a role in the
regulation of RNA binding activity of ferritin in vivo,
extracts were prepared from 48-h cultured rat hepatocytes treated for
2 h with glucagon and thereafter in addition for another 3 h
with insulin in the absence and presence of okadaic acid. Ferritin in
cytosolic extracts from okadaic acid-treated cells, irrespective
whether treated simultaneously with insulin or not, possessed lower
binding activity to the PCK1 transcript than protein from non-okadaic
acid-treated cells (Fig. 7). This implies
also that in vivo phosphorylation of the protein decreased and dephosphorylation increased binding to the PCK mRNA. Ongoing gene transcription did not play a role in the regulation of protein binding to PCK1 transcript, because addition of the transcriptional inhibitor actinomycin D to the hepatocytes did not affect formation of
complex 1. The half-life time of ferritin in the liver was determined
at 50-70 h (27). Therefore, it is unlikely that under the chosen
experimental conditions, i.e. preparation of extracts 3 h after application of insulin, okadaic acid, or actinomycin D, changes
in binding activity were due to changes in ferritin content of the
hepatocytes. It can be assumed that the decrease in protein binding to
PCK1 transcript was rather due to the decrease in the affinity or
activity of ferritin binding to RNA.
Iron Decreased Cytosolic Protein Binding to RNA in Vitro
Ferritin is the iron storage protein in the liver. This
might indicate that complex formation with the purified protein and RNA
can be regulated by iron. Therefore the gel retardation assay with the
PCK1 transcript was performed with purified rat liver ferritin
(Superdex column fractions 36-38) after preincubation for 30 min at
37 °C with increasing concentrations of iron or the iron chelating
agent desferal (Fig. 8). Increasing iron
concentration attenuated binding of ferritin to the PCK1 transcript in
a concentration-dependent fashion. Yet, desferal did not
affect protein binding to the PCK1 transcript.
The expression of PCK is in
part regulated at the level of mRNA degradation (13, 18, 19). To
find regulatory proteins which might be involved in this process, the
3-UTR of PCK mRNA was used as target for the selection of
RNA-binding proteins from rat liver. By this procedure a RNA-binding
protein was purified and identified as rat ferritin light chain. The
RNA-protein complex formed not only with transcripts containing PCK
mRNA 3
-UTR sequences but with a variety of different mRNA
3
-UTR species (Fig. 2). Computer alignment with these different RNAs
revealed no sequence homologies, which could be the common target
sequence for protein binding. This indicates that complex, so far
unidentified, RNA structures might be necessary for RNA-protein complex
formation. Also searching the SWISS protein data bank for consensus
peptide motifs in ferritin, which could be putative contact sites for RNA, revealed no similarities with known RNA-binding peptide motifs, e.g. with the RNP motifs in heterogeneous nuclear
ribonucleoproteins (28). However, it is clear that ferritin functions
as a RNA-binding protein with the potency to bind to a broad range of
different RNA species.
RNA binding properties of ferritin and ferritin-homologs have already been described. A prosome-like particle, which was believed to play a role in protein degradation, was isolated and turned out to be composed of identical apoferritin subunits. This particle was associated with small RNA molecules, probably various tRNA species (24). These RNAs were capable of hybridizing with globin mRNA (24, 29). From Artemia cysts the protein artemin was purified, which shared homology with vertebrate ferritins and probably functions as iron storage protein (30). In addition, artemin belongs to a class of RNA-binding proteins and was proposed to maintain the integrity of the dehydrated cyst (31, 32).
Ferritin is the iron storage protein in liver, which protects cells against cytotoxic effects of reactive oxygen species derived from iron-catalyzed free radical reactions (for review, Ref. 33). Three different types of ferritin were described (apoferritins), the L (light) and two variants of H (heavy) chains, of which the L type is prevalent in rat liver. The apoferritins form a holoferritin complex, which consists of 24 subunits forming a very stable shell for the storage of about 4500 iron ions as FeIIIO(OH) inside the complex (34).
Early experiments using transcriptional inhibitors showed that after an iron challenge the increase in cellular ferritin concentrations were not due to an increase in gene transcription but rather to the increase in the translation of ferritin mRNA (23). Under normal, non-iron depleted conditions ferritin concentrations remain fairly constant because of the stability of ferritin, which is reflected by the long half-life time of 50-70 h (27). Hence, the changes in binding activity of ferritin to PCK1 transcript observed in the present study both in vitro (Fig. 6) and in the experiments using cultured hepatocytes (Fig. 7) were not due to changes in protein content but either to alterations in the binding affinity of ferritin or to changes in the concentration of active RNA-binding protein.
Translation of ferritin mRNA is controlled by iron-regulated
RNA-protein interactions. The iron responsive element (IRE) was identified in the 5-UTR of ferritin mRNA comprising sequences capable of forming a hairpin loop, which is recognized by the iron
responsive element-binding protein (IRP). Binding of IRP is highly
regulated by iron. Under iron deficiency the protein occupies the IRE
so that the ferritin mRNA can no longer be translated. Under iron
excess IRP complexes Fe2+ causing conformational changes in
the IRP, which in this state no longer binds to the IRE, so that the
ferritin mRNA can be translated. The Fe2+-loaded IRP
functions as cytosolic aconitase (35).
Dephosphorylation enhanced and phosphorylation attenuated formation of the RNA-protein complex between ferritin and PCK1 transcript. Anti-phosphoserine or anti-phosphothreonine antibodies attenuated complex formation, whereas an anti-phosphotyrosine antibody did not interfere (Fig. 6). This suggested that phosphorylated serine and threonine but not tyrosine residues are located in close proximity to the RNA-binding site of ferritin. Indeed two potential phosphorylation sites for the serine/threonine kinases casein kinase II (peptides TEVE and SQDE) and for protein kinase C (PKC; SAR) are present in ferritin. However, the function of these sites in the regulation of RNA-protein complex formation remains to be established. Phosphorylation of serine residues in ferritin has already been described earlier (36).
In cultured rat hepatocytes the degradation of PCK mRNA was accelerated by insulin and abolished in the presence of the phosphatase inhibitor okadaic acid.2 Whereas treatment of cultured rat hepatocytes with insulin did not change RNA-protein complex formation, treatment with okadaic acid diminished complex formation (Fig. 7). This appears to show that ferritin binding to PCK mRNA was not regulated in vivo by insulin but obviously by okadaic acid-influenced phosphorylation/dephosphorylation. The diminution of complex formation in the presence of okadaic acid and the inhibition of PCK mRNA decay by okadaic acid might indicate a functional correlation between ferritin binding and mRNA degradation. In this hypothetical model dephosphorylated ferritin would function as a stimulator of PCK mRNA degradation.
In FTO-2B rat hepatoma cells cAMP stabilized PCK mRNA against
degradation (18, 19). A cytosolic protein had been identified, which
bound specifically to different 30-mers of the PCK mRNA 3-UTR
in vitro (22). Protein binding was diminished after
phosphorylation of the protein and after treatment of FTO-2B rat
hepatoma cells with cAMP. Conversely, dephosphorylation after in
vitro treatment of the cytosolic proteins by alkaline phosphatase
enhanced RNA-protein complex formation (22). In the present study
binding of ferritin to RNA was also diminished after phosphorylation
and augmented after dephosphorylation. However, the protein described
in Ref. 22 is very likely different from ferritin because of various biochemical differences. The binding activity of the protein from FTO-2B hepatoma cells was increased by KCl, it bound to specific sequences in the 3
-UTR of PCK mRNA, partial purification lead to
the loss of an inhibitory activity and finally the molecular mass was
determined at 100 kDa.
Evidence increases that cellular signal transduction pathways involving phosphorylation and dephosphorylation cycles play an important role in the control of cytosolic mRNA metabolism like mRNA degradation (for review, see Ref. 37). Various different mRNAs were destabilized in T-cells (38) but granulocyte macrophage-colony stimulating factor mRNA was stabilized in thymoma cells (39) by protein kinase C activation with phorbol ester. Phosphorylation following serine/threonine phosphatase inhibition by okadaic acid diminished C heterogeneous nuclear ribonucleoprotein binding to pre-mRNA (40). IRE binding activity was increased by phosphorylation of the IRE-binding protein by protein kinase C (41).
Functional Significance of Ferritin as RNA-binding Protein1 g of liver contains a cytoplasmic hepatocellular volume of 0.7 ml and 1.5 nmol of ferritin (see above and Ref. 27). Therefore, the ferritin level in hepatocytes is 1.5 nmol/0.7 ml = 2.1 µM. In the assay PCK1 transcript was half-saturated at 0.12 µM ferritin (cf. "Results"), which is more than 10-fold lower than the total cellular ferritin concentrations. However, the total ferritin level is probably clearly higher than that freely available for RNA binding. Because ferritin is associated with different cellular compartments, which are not freely interconvertable, it cannot exactly be defined, which is the level of ferritin employed in iron storage or in other cellular functions (27). The half-saturation concentration determined may be in the range of "free" ferritin. Therefore, it is feasible that binding of ferritin to RNA is of physiological relevance.
Some evidence exists that ferritin via its RNA-binding capacity might
play a role in the general metabolism of RNA. For example, addition of
ferritin-containing prosome-like particles to an in vitro
translation system inhibited the translation of globin mRNA, indicating that ferritin functioned as a translational repressor (24,
29). This had also been suggested in earlier reports, which claimed
that ferritin bound to the 5-UTR of its own mRNA and stored the
mRNA in a translational incompetent form (42). Due to its similar
amino acid composition and immunological cross-reactivity the
RNA-binding protein from Artemia cysts had been suggested to
be related to the elongation factor eEF-Ts, which, however, remains to
be experimentally established (31). These data suggest that ferritin,
besides its function as iron storage protein, may not have a unique but
rather a general function in the regulation of RNA metabolism at
various levels (23, 24).
The present study had the goal to identify protein(s), which might be involved in the regulation of PCK mRNA degradation. Ferritin was isolated and its RNA binding characteristics determined (Figs. 1, 2, 3, 4). Because no specific binding to PCK mRNA but rather a general binding to mRNAs was detected, it seems likely that ferritin is not only involved in PCK mRNA metabolism but in general processes, which, however, at present cannot be defined. It might be relevant that in the present study RNA-protein complex formation was inhibited by iron (Fig. 8). This might link the iron loading status of the cell to the regulation of general RNA degradation.
We are greatly indebted to Drs. H. Jäckle and P. C. Heinrich who critically read the manuscript and gave helpful advise in preparing the manuscript. We thank Drs. B. Schmidt and T. Selmer for invaluable help with the sequencing of the purified protein.