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Endocrinology Vol. 148, No. 1 252-257
Copyright © 2007 by The Endocrine Society

Thyroid Hormones Induce Sumoylation of the Cold Shock Domain-Containing Protein PIPPin in Developing Rat Brain and in Cultured Neurons

Epifania Bono, Vincenza Compagno, Patrizia Proia, Lavinia Raimondi, Gabriella Schiera, Vincenzo Favaloro, Valerio Campo, Maria Donatelli and Italia Di Liegro

Dipartimento di Biologia Cellulare e dello Sviluppo ‘Alberto Monroy’ (E.B., L.R., V.F., V.C.), Dipartimento Biomedico di Medicina Interna e Specialistiche (V.C., M.D.), Dipartimento di Neurologia, Oftalmologia, Otorinolaringoiatria e Psichiatria (P.P.), and Dipartimento di Scienze Biochimiche (G.S., I.D.L.), University of Palermo, 90127 Palermo, Italy

Address all correspondence and requests for reprints to: Italia Di Liegro, Dipartimento di Scienze Biochimiche, Via del Vespro 129, 90127 Palermo, Italy. E-mail: diliegro{at}unipa.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We previously identified a cold shock domain (CSD)-containing protein (PIPPin), expressed at high level in brain cells. PIPPin has the potential to undergo different posttranslational modifications and might be a good candidate to regulate the synthesis of specific proteins in response to extracellular stimuli. Here we report the effects of T3 on PIPPin expression in developing rat brain. We found that a significant difference among euthyroid and hypothyroid newborn rats concerns sumoylation of nuclear PIPPin, which is abolished by hypothyroidism. Moreover, T3 dependence of PIPPin sumoylation has been confirmed in cortical neurons purified from brain cortices and cultured in a chemically defined medium (Maat medium), with or without T3. We also report that about one half of unmodified as well as all the sumoylated form of PIPPin could be extracted from nuclei with HCl, together with histones. Moreover, this HCl-soluble fraction remains in the nucleus even after treatment with 0.6 M KCl, thus suggesting strong interaction of PIPPin with nuclear structures and perhaps chromatin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
THE POSTTRANSCRIPTIONAL fate of eukaryotic mRNA depends on association with different families of RNA-binding proteins (RBPs) into ribo-nucleoprotein particles that can be anchored to a variety of cell structures (for review, see Refs. 1, 2, 3, 4). By recognizing and binding unifying features, present on transcripts encoded by genes to be coordinately expressed, RBPs can regulate stability, localization and translation of specific sets of mRNAs, in response to extracellular stimuli, as though they formed posttranscriptional operons (5).

Cold shock domain (CSD)-containing proteins, also called Y-box-proteins, form a highly conserved family of RBPs present in all organisms, from bacteria to humans, with single-stranded (ss) DNA and/or ssRNA-binding activity (6, 7, 8, 9). Several members of the family have been suggested to couple nuclear history of a transcript to its translational fate (10, 11, 12, 13).

The critical phase of rat brain maturation depends strictly on thyroid hormone (T3): in fact, both hypo- and hyperthyroid animals suffer severe and irreversible abnormalities in the central nervous system. In the fetal life, thyroid hormones are produced by the mother and even moderate and transient maternal hypothyroidism can alter brain organization of the fetuses (14, 15). Although this dependence has been known for a long time (see, for instance, Refs. 16, 17, 18, 19), the molecular mechanisms through which these hormones act on developing brain are not completely understood.

Several years ago, we observed that T3 can induce in rat cortical neurons, cultured in a chemically defined medium (Maat medium; Ref. 20), the overall reorganization of chromatin that characterizes terminal differentiation of cortical neurons in vivo (21, 22). More recently, we described a CSD-containing protein that seemed able to bind mRNAs encoding histone variants (23, 24, 25) and is present both in nucleus and in the cytoplasm of brain cells (24). Because other CSD-containing proteins have the ability to interact both with RNA and chromatin, we investigated the possibility that PIPPin binds to chromatin. We also looked for effects of T3 on PIPPin expression by comparing newborn euthyroid rats with newborns delivered by rats treated with 6-propyl-2-thiouracyl (PTU) since the last week of pregnancy. We analyzed in parallel a primary culture system in which rat cortical neurons were purified from brain cortices and cultured on laminin in a chemically defined medium, with or without T3, as previously described (22).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Experimental animals
Winstar rats and New Zealand female rabbits (Stefano Morini, San Polo d’Enza, Italy) were housed in the institutional animal care facility of the Department of "Biologia Cellulare e Sviluppo" (University of Palermo, Palermo, Italy), under direction of a licensed veterinary who approved protocols. Procedures involving animals were conducted according to the European Community Council Directive 86/609, OJL 358 1, 12 December 1987. The number of animals used has been minimized as well as their suffering.

Induction of hypothyroidism
Female rats were treated with 200 mg/liter of PTU in drinking water from d 14 of gestation throughout lactation. Concentration of circulating T4 was determined in both adult and newborn rats by RIA (Animaldx; Diagnostic Systems Laboratories, Inc., Webster, TX). As detailed in the previous section, the whole procedure was performed under direction of a licensed veterinary who approved protocols.

Preparation of tissue extracts
Fresh brain tissue from either adult or newborn rats was prepared as previously described (26). Briefly, cortical hemispheres were homogenized in nuclei buffer [0.32 M sucrose; 50 mM sodium phosphate buffer (pH 6.5); 50 mM KCl; 0.5 mM spermine; 0.15 mM spermidine; 2 mM EDTA, and 0.15 mM EGTA], containing the protease inhibitors aprotinin (2 µg/ml), antipain (2 µg/ml), leupeptin (2 µg/ml), pepstatin A (2 µg/ml), benzamidine (1.0 mM), and phenylmethylsulfonyl fluoride (1.0 mM), all purchased from Sigma-Aldrich (Milano, Italy). The homogenate was centrifuged at 1000 x g for 10 min at 4 C: the supernatant (postnuclear extract) was split into aliquots and rapidly frozen, whereas the nuclear pellet was washed once in reticulocyte-buffered saline [RSB: 10 mM NaCl; 10 mM Tris-HCl, (pH 7.5); 1.5 mM MgCl2] and resuspended in 1 vol RSB. After adding 1 vol of high-salt solution (RSB, containing 1.6 M KCl), samples were incubated for 30 min on ice and centrifuged at 10,000 x g for 10 min, at 4 C. The supernatant (nuclear extract) was split into aliquots and frozen. The pellets (that contain DNA as well as proteins tightly associated with DNA), or alternatively total nuclei, were resuspended in RSB, containing 1.0 mM phenylmethylsulfonyl fluoride, and HCl was added at a final concentration of 0.4 N, to obtain HCl-soluble basic proteins as described by Castiglia et al. (27). After incubation for at least 4 h, at 4 C, acid-soluble material was pelletted at 10,000 x g for 20 min and the acid-soluble proteins were recovered from the supernatant by precipitation with 10 volumes of acetone at –20 C and centrifugation at 10,000 x g for 20 min. HCl-soluble proteins were finally resuspended in distilled water, split into aliquots and frozen.

Neuronal cultures and extracts
Neurons were prepared from embryonic d 16 rat cerebral cortices, as described in detail elsewhere (20, 28). Cells were cultured for 10 or 15 d in a selective, serum-free medium (Maat medium), on tissue culture dishes (Diafarm Union srl, Catania, Italy), precoated with laminin (Sigma-Aldrich), with or without 2.0 x 10–8 M T3. To obtain postnuclear-, nuclear-, and HCl-soluble proteins, cells were collected by centrifugation at 400 x g for 3 min, washed once with PBS and processed as described above for the preparation of fresh brain tissue extracts.

Preparation of recombinant proteins for antibody production
Recombinant PIPPin with an N-terminal tag of six histidines (6His-PIPPin; Ref. 29) was expressed in BL21-SI Escherichia coli cells and purified by metal-affinity chromatography on a selective nickel-nitriloacetic (Ni-NTA)-conjugated agarose matrix (QIAexpress System, QIAGEN spa, Milano, Italy). Purified protein was used to immunize New Zealand female rabbits (Stefano Morini, San Polo d’Enza, Italy), as already described (24). Specificity and titer of antisera, obtained after several boosts, were tested by Western blot.

Northern and Western analyses
Total RNA from either fresh brain tissue or purified neurons was isolated according to Chomczynsky and Sacchi (30) and separated by electrophoresis on 1.5% agarose, 6% formaldehyde gels. RNA was then transferred to nylon membrane (Hybond, Amersham, Little Chalfont, UK) and hybridized to 32P-labeled PIPPin probe, as already described (23).

Proteins (15–30 µg of total or fractionated cell extracts) were separated by electrophoresis on denaturing 15% polyacrylamide slab gels (SDS-PAGE). HCl-soluble nuclear proteins (150–200 µg) were also separated by two-dimensional electrophoretic analysis, with isoelectrophocusing, in the 5–8 pH range, in the first dimension, and SDS-PAGE in the second dimension. Proteins from both mono- and two-dimensional gels were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Billerica, MA) in 0.1 M 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS; Sigma-Aldrich), pH 11; 10% Methanol, at 170 mA for 45 min, at 4 C. Membranes were probed with the following antibodies: 1) home-made rabbit polyclonal anti-PIPPin serum; 2) mouse monoclonal antiubiquitin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 3) rabbit polyclonal anti-SUMO-1 (small ubiquitin-like modifier 1) antibodies (Santa Cruz Biotechnology, Inc.). Secondary antibodies were alkaline phosphatase-conjugate antirabbit or antimouse IgGs (Promega Corp., Madison, WI).

Immunofluorescence
Neurons were cultured on laminin-coated coverslips for 10–15 d. Cells were then fixed with 96% ethanol, on ice, for 10 min and then permeabilized for 5 min with 0.1% Triton X-100, in PBS. Cells were then incubated with the following antibodies: 1) home-made rabbit polyclonal anti-PIPPin serum; 2) rabbit polyclonal anti-SUMO-1 antibodies (Santa Cruz Biotechnology, Inc.); or 3) mouse monoclonal anti-growth-associated protein 43 (GAP43; Roche Diagnostics, Monza, Italy) antibodies. The secondary antibodies were antirabbit- or antimouse-IgG, conjugated to fluorescein isothiocyanate or to rhodamine (tetramethyl rhodamine iso-thiocyanate; Promega Corp.). Cells were finally stained for DNA by treating them with Vectashield mounting medium for fluorescence, containing 4',6-diamidino-2-phenylindole (Vector Laboratories, Youngstown, OH). Cells were observed in an Olympus BX-50 microscope (Olympus Italia s.r.l., Segrate, Italy) equipped with Vario Cam B/W camera (Nikon Instruments s.p.a., Calenzano, Italy), and elaborated by image-pro/plus Media Cybernetics (Gleichen, Germany).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Induction of hypothyroidism in female and newborn rats
The critical phase of brain maturation clearly depends on thyroid hormones. However, the molecular mechanisms responsible for this dependence are not completely understood. Only a few genes have been indeed recognized as direct targets of thyroid hormones in the brain, among which GAP 43 (also known as neuromodulin; Ref. 31), basic transcription element-binding protein (32), nuclear pore glycoprotein (32), and bone morphogenetic protein-4 (32). In addition to the traditional T3 genomic action, dynamic nongenomic actions have been also suggested (33, 34).

At the beginning of terminal differentiation, neurons are definitively postmitotic, but nevertheless undergo dramatic modifications of chromatin overall organization (21, 22), probably dependent on synthesis and incorporation into chromatin of replication-independent histone variants (35, 36, 37, 38, 39). Regulation of the synthesis of histone variants in developing rat brain is largely controlled at posttranscriptional level (40) and possibly involves different classes of RBPs (23, 24, 26), among which PIPPin, a Y-box protein present both in the nucleus and the cytoplasm of specific classes of brain cells (24, 25). Our working hypothesis was that brain maturation involves modifications of the concentration and/or the activity of nucleic acid-binding regulatory factors such as PIPPin. In looking for extracellular candidates able to regulate these events, we started with thyroid hormones for at least three reasons. First of all, as mentioned, these hormones are clearly involved in maturation of brain, where they affect late events such as cell migration, terminal differentiation of both glial and neuronal cells, and synaptogenesis (19). Second, several years ago, we observed that thyroid hormones are involved in the overall rearrangement of neuronal chromatin in culture (21). Third, pyramidal cells of the neocortex and the Purkinjie cells of the cerebellum, that are among the cells more strongly affected by hypothyroidism (19), are the same cells that express PIPPin (25).

In the first phase of our research, we induced hypothyroidism in pregnant and newborn rats by treating female rats with 6-PTU since the second week of gestation and during lactation. This protocol was effective in inducing hypothyroidism in the newborns. As shown in Table 1Go, indeed, T4 concentration in the blood of control and PTU-treated female, as well as newborn, rats differed significantly (P < 0.001 at all the stages).


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  		TABLE 1. T4 concentration in sera from control and PTU-treated rats (nanomoles/liter)

 
Effects of T3 on PIPPin expression in developing brain
To investigate the possible correlation between T3 treatment and PIPPin expression, we first studied distribution of PIPPin mRNA in developing brain of eu- and hypothyroid newborn rats. Equal amounts of total RNA, purified from treated and untreated rats at different days after birth, were analyzed by Northern blot, using a PIPPin-specific probe that, as expected, revealed a band of about 2.5 kb, in all the samples analyzed (not shown). The results obtained from at least three Northern analyses were used to draw the histograms reported in Fig. 1Go. As shown, PIPPin mRNA tends to be more concentrated in hypothyroid rats where it reaches a significantly higher (P = 0.013) level at d 15 after birth (Fig. 1Go). However, the higher concentration of mRNA in hypothyroid rats does not correspond to significantly modified levels of PIPPin protein in the postnuclear fraction (Fig. 2AGo), or in the protein fraction extracted from nuclei with 0.6 M KCl (nuclear extract) (Fig. 2BGo). In this latter fraction, a significant difference (P = 0.049) was only found at the postnatal d 5.


Figure 1
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  		FIG. 1. Expression of PIPPin mRNA in euthyroid (eu) (gray bars) and hypothyroid (hy) rats (white bars) in the first 2 wk of postnatal development. Graphic representation of statistical analysis of Northern data (mean of three independent analyses). Bars indicate mean values for each group of samples. SDs are also indicated. Total RNAs were prepared from brains of 0-d-(P0), 5-d-(P5), 10-d-(P10), and 15-d-(P15) old newborn rats and hybridized to a 32P-labeled PIPPin probe (22 ).

 

Figure 2
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  		FIG. 2. Concentration of PIPPin in different brain subcellular fractions at different stages of postnatal development. A and B, Graphical representation of statistical analysis of Western data (mean of at least three independent analyses), obtained from either the postnuclear fraction (A) or the nuclear salt extract (B), of euthyroid (eu) (gray bars) and hypothyroid (hy) rats (white bars). Bars indicate mean values for each group of samples. SDs are also indicated. C, Typical distribution of PIPPin (~28 kDa) in different subcellular fractions, from euthyroid newborn rats at d 5 of postnatal development (P5). D, Representative example of western analysis of PIPPin, showing the bands immunostained by anti-PIPPin antibodies in the HCl-soluble nuclear fraction (about 20 µg per sample) from eu- and hypothyroid newborn rats, at different stages of development. Subcellular fractions were prepared, as described in Materials and Methods, from brains of 0-d-(P0), 5-d-(P5), 10-d-(P10), and 15-d-(P15) old newborn rats. Nex, Nuclear salt-extract; PN, postnuclear fraction; HCl, nuclear HCl-soluble fraction.

 
When we treated with HCl the nuclear pellet left behind by salt extraction, we obtained an acid-soluble fraction that still contains PIPPin. The same result was obtained when nuclei were directly extracted with HCl: a significant proportion of PIPPin was found to be acid soluble (Fig. 2CGo). More interestingly, at d 15 of postnatal development, anti-PIPPin antibodies recognized, in the acid-soluble fraction, and only in euthyroid brain, a slower migrating band of about 35 kDa (Fig. 2DGo, signal indicated by the arrow).

Effects of T3 on PIPPin sumoylation in vivo
We previously noticed by immunofluorescence that a high proportion of intranuclear PIPPin showed a peripheral localization that suggested an anchorage to nuclear structures (24). Because specific localization of nuclear proteins has been often reported to require posttranslational modifications such as ubiquitination or sumoylation, we reasoned that the protein with higher apparent mass, recognized by anti-PIPPin serum, could be ubiquitinated or sumoylated PIPPin. Therefore, we probed HCl-soluble nuclear proteins with antiubiquitin and anti-SUMO1 antibodies. Antiubiquitin antibodies did not recognize any protein with apparent mass of 35 kDa. On the contrary, anti-SUMO1 antibodies not only recognized a protein of 35 kDa but identified such a protein only in euthyroid rats (Fig. 3AGo). To be sure that the 35-kDa bands evidenced by anti-PIPPin and anti-SUMO1 antibodies were the same protein, we also performed two-dimensional analysis. As shown in Fig. 3BGo, there is again a clear correspondence between the slower spot recognized by anti-PIPPin antibodies and one spot recognized by anti-SUMO1 antibodies. We concluded that PIPPin can be sumoylated and that this modification is under the control of thyroid hormones.


Figure 3
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  		FIG. 3. Western analysis of PIPPin present in the HCl-soluble nuclear fraction from rats at d 15 of postnatal development (P15), after mono- (A) or two-dimensional electrophoretic analysis (B). In the monodimensional analysis, twin samples of acid-soluble proteins (about 20 µg each) from either hypo- (Hy) or euthyroid (Eu) animals were separated by electrophoresis and blotted onto PVDF membrane. The membrane was then cut into two halves, each of which was incubated with either anti-PIPPin- (a) or anti-SUMO1 (b) antibodies. In the two-dimensional analysis, HCl-soluble twin samples (about 50 µg each) from euthyroid rat brain at d 15 of postnatal development (P15) were separated by isoelectrofocusing (IEF), in the 5–8 pH range, in the first dimension, and by 15% SDS-PAGE in the second dimension. The samples were then blotted onto PVDF membranes. Each membrane was incubated with either anti-PIPPin- (a) or anti-SUMO1 (b) antibodies. SDS, Sodium dodecyl sulfate.

 
Effects of T3 on PIPPin expression in isolated cortical neurons
In developing brain many different populations of nerve as well as glial cells are present. This fact should hamper interpretation of the functional meaning of PIPPin sumoylation. We therefore analyzed the effect of T3 in a culture system in which neurons are purified from rat cortical hemispheres and cultured for 10–15 d in a serum-free, chemically defined medium, in the presence or not of T3 (2.0 x 10–8 M; Ref. 21). Also, in this case we did not find significant differences in the concentration of PIPPin when postnuclear and nuclear extracts of control and T3-treated cells were compared (not shown). However, a 35-kDa protein was clearly visible in the acid-soluble fraction from neurons treated with T3 (T10 and T15, Fig. 4AGo). This band was also recognized by anti-SUMO1, but not by antiubiquitin, antibodies (Fig. 4BGo). In conclusion, we can say that T3 influences PIPPin sumoylation both in vivo and in culture.


Figure 4
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  		FIG. 4. Western analysis of PIPPin isoforms present in the HCl-soluble nuclear fractions (about 20 µg per sample) from rat cortical neurons cultured for 10 or 15 d, without- (C) or with- (T) T3. A, Immunostaining of HCl-soluble proteins with anti-PIPPin antibodies; B, immunostaining of HCl-soluble proteins with three different antibodies. Identical couples of samples were separated on the same gel and blotted onto PVDF membrane. The membrane was then cut into three pieces, each of which was incubated with anti-PIPPin-, antiubiquitin-, or anti-SUMO1- antibodies. P, PIPPin; sP, sumoylated PIPPin. Marker sizes (in kilodaltons) are reported on the left margin, for reference.

 
Nuclear localization of PIPPin
We finally tested by immunofluorescence PIPPin localization in neurons cultured with or without T3 (Fig. 5Go). These experiments showed that a higher proportion of nuclear PIPPin has a peripheral localization in T3-treated cells (Fig. 5Go, D and J) than in control neurons (Fig. 5Go, A and G): see the fluorescent rings indicated by arrows. In parallel, cells were immunostained with antineuromodulin (GAP-43: Fig. 5Go, B and E), an already known target of T3 in neurons (31, 34). T3-treated neurons (Fig. 5EGo) show more intensively stained neurites respect to untreated cells (Fig. 5BGo). Cells were also probed with anti-SUMO-1 antibodies (Fig. 5Go, H and K). Interestingly, SUMO-1 shows a nuclear peripheral localization resembling that of PIPPin: compare the fluorescent rings, indicated by arrows, in Fig. 5Go, H and K, with those of Fig. 5Go, D and J.


Figure 5
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  		FIG. 5. Intracellular localization of PIPPin in neurons cultured without T3 (A–C, and G–I) or with T3 (D–F, and J–L), for 2 wk, on laminin-coated coverslips. After fixation and permeabilization, cells were treated with the following antibodies: 1) rabbit anti-PIPPin antibodies (A, D, G, and J); 2) mouse monoclonal anti-GAP43 antibodies (B and E); 3) rabbit polyclonal anti-SUMO-1 antibodies (H and K). The secondary antibodies were antirabbit IgG conjugated to fluoresceine (A, D, G, and J) or antimouse- or antirabbit-IgG, conjugated to rhodamine (B, E, H, and K). To stain nuclei, cells were also treated with 4',6-diamidino-2-phenylindole (C, F, I, and L). Cells were observed in an Olympus BX-50 microscope equipped with Vario Cam B/W camera, and elaborated by image-pro/plus Media Cybernetics. The arrows indicate regions, at the periphery of nuclei, in which PIPPin and SUMO-1 appear as brilliant rings. Each row reports the same field immunostained with different antibodies. SDS, Sodium dodecyl sulfate.

 
In conclusion: 1) in the nuclear acid-soluble fraction from euthyroid P15 brain, a 35-kDa protein was evident that was not found in hypothyroid rats; 2) on the basis of both one- and two-dimensional immunoblots, the 35-kDa protein was suggested to be a sumoylated form of PIPPin; 3) sumoylated PIPPin is also present in nuclei from cortical neurons cultured in a chemically defined medium containing T3, whereas it was not found in neurons cultured in the absence of hormone. This latter finding confirmed T3 dependence of PIPPin sumoylation.

Thus, which might be the function of this modification? On the basis of two-dimensional electrophoresis, we previously deduced that PIPPin is present in the nucleus in two different forms: one basic and one acidic (24). We now report that about one half of PIPPin can be indeed extracted with HCl (the basic fraction), whereas another half remains in the acid-insoluble pellet. This latter fraction is the same fraction that can be extracted from nuclei with salt: after salt-extraction, indeed, we can still recover from nuclei about one half of PIPPin by HCl. But, salt extraction recovers in general proteins less firmly bound to DNA. If basic PIPPin remains behind, we have to conclude that it strongly binds to chromatin, perhaps to DNA: PIPPin is indeed a CSD-containing protein and many proteins of this family are transcriptional regulators. We previously reported evidence that PIPPin is an RNA-binding factor and that only acidic PIPPin, present in the nuclear salt-extract, is able to bind RNA (24). This latter fraction has been suggested to be phosphorylated (24). PIPPin has indeed several putative kinase recognition sites, some of which have been recently shown to undergo phosphorylation (41).

In this context, acidic PIPPin might be involved with binding and export to the cytoplasm of mRNA, whereas sumoylation of a significant number of PIPPin molecules could drive localization of PIPPin activity to specific nuclear domains, where it might participate in earlier stages of mRNA metabolism. Herein, a specific localization of PIPPin, in the form of rings at the periphery of the nucleus, was also suggested by fluorescence microscopy.

To our knowledge, this is the first report that suggests a role of thyroid hormones in sumoylation and localization of a nuclear protein, and experiments are now in progress to investigate the mechanisms through which this role is played.


   Acknowledgments
 
We thank C. Di Liegro for helpful discussions.


   Footnotes
 
This work was supported by the Italian Ministero dell’Istruzione e della Ricerca (MIUR) and by the University of Palermo (Università degli Studi di Palermo), Italy. E. Bono, V. Compagno, and P. Proia were supported by Ph.D. fellowships of the Università degli Studi di Palermo; L. Raimondi and G. Schiera were supported by postdoctoral fellowships of the Università degli Studi di Palermo.

Disclosure Statement: The authors have nothing to declare.

First Published Online October 19, 2006

Abbreviations: CSD, Cold shock domain; GAP, growth-associated protein; PTU, 6-propyl-2-thiouracyl; PVDF, polyvinylidene difluoride; RBP, RNA-binding proteins; RSB, reticulocyte-buffered saline; ss, single stranded; SUMO-1, small ubiquitin-like modifier 1.

Received May 16, 2006.

Accepted for publication October 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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