RNA association or phosphorylation of the RS domain prevents aggregation of RS domain-containing proteins

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Biochimica et Biophysica Acta 1780 (2008) 214 – 225 www.elsevier.com/locate/bbagen

RNA association or phosphorylation of the RS domain prevents aggregation of RS domain-containing proteins Eleni Nikolakaki a , Victoria Drosou a , Ioannis Sanidas a , Philippos Peidis a , Thomais Papamarcaki b,c , Lilia M. Iakoucheva d , Thomas Giannakouros a,⁎ a

Laboratory of Biochemistry, Department of Chemistry, The Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece b Laboratory of Biological Chemistry, Medical School, The University of Ioannina, 451 10 Ioannina, Greece c Foundation for Research and Technology-Hellas/Biomedical Research Institute 451 10 Ioannina, Greece d Laboratory of Statistical Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 25 July 2007; received in revised form 11 October 2007; accepted 18 October 2007 Available online 1 November 2007

Abstract Domains rich in alternating arginine and serine residues (RS domains) are found in a large number of eukaryotic proteins involved in several cellular processes. According to the prevailing view RS domains function as protein interaction domains, thereby promoting the assembly of higher-order cellular structures. Furthermore, recent data demonstrated that the RS regions of several SR splicing factors directly contact the premRNA in a nonsequence specific but functionally important fashion. Using a variety of biochemical approaches, we now demonstrate that the RS domains of three proteins, not directly associated with the splicing reaction, such as lamin b receptor, acinus and peroxisome proliferator-activated receptor gamma coactivator-1 alpha, associate mainly with nuclear RNA and that this association is conducive in retaining the proteins in a soluble form. Phosphorylation by SRPK1 prevents RNA association, yet it greatly increases the fraction of the proteins recovered in soluble form, thereby mimicking the RNA effect. Based on these results we propose that the tendency to self-associate and form aggregates is a general property of RS domain-containing proteins and could be attributed to their disordered structure. RNA binding or SRPK1-mediated phosphorylation prevents aggregation and may serve to modulate the RS domain interaction modes. © 2007 Elsevier B.V. All rights reserved. Keywords: RS domain; LBR; Acinus; PGC-1a; SRPK1; Intrinsic disorder

1. Introduction Originally identified in three genetically defined Drosophila splicing regulators, transformer, transformer-2 and suppressor of white apricot, RS domains were considered a new class of targeting signals, directing concentration of proteins in a subnuclear compartment implicated in the splicing process [1]. In the following years, numerous RS domain-containing proteins were identified that are functionally associated with a number of Abbreviations: RS, arginine serine; LBR, lamin B receptor; PGC-1a, peroxisome proliferator-activated receptor gamma coactivator-1 alpha; SRPK1, SR protein-specific kinase 1; RRM, RNA recognition motif; HP1, Heterochromatic protein 1 ⁎ Corresponding author. Laboratory of Biochemistry, Department of Chemistry, The Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece. Tel.: +1 30 2310 997702; fax: +30 2310 997689. E-mail address: [email protected] (T. Giannakouros). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2007.10.014

cellular processes, in addition to pre-mRNA splicing, such as chromatin structure, transcription by RNA polymerase II, cell cycle, cell structure and germ cell development. [for review see Ref. [2]]. RS domain proteins can be subdivided into two groups: (a) those that contain one or two RNA recognition motifs (RRMs) in addition to the RS domain and (b) those that do not contain an RRM. Serine-arginine rich proteins (SR proteins), mediating both constitutive and alternative splicing of pre-mRNAs, are the most well-known proteins of the first group. Although RS domains clearly function as splicing-activation domains [3], the mechanism by which they do so is still controversial. According to the protein-protein interaction view the RS domain of an enhancer bound SR protein interacts directly with other splicing factors that also contain an RS domain, in a phosphorylation-dependent manner, and, thus, facilitate recruitment of the spliceosome [4,5]. On the other hand, several reports documented the ability of RS

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domains to participate in functionally important, nonsequence specific interactions, with pre-mRNA, thereby favouring a different model of RS domain function. More specifically, the RS region of the large subunit of U2AF was shown to interact with the pre-mRNA branch site in a way that facilitates base-pairing with U2 snRNA [6]. Furthermore, recent data demonstrated that RS domains of several different splicing factors sequentially interact with the branchpoint and the 5′ splice site within the mRNA, and, thus, promote the splicing reaction [7–9]. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARγ coactivator-1α, PGC-1a) and Acinus also belong to the first group of RS domain proteins. PGC-1α can bind to and coactivate most members of the nuclear receptor family [reviewed in Ref. [10]]. The C-terminal region of PGC-1a, that harbors an RS domain and an RNA binding domain, is critical for its transcriprtional activity [11]. In addition, the same region was reportedly required for targeting PGC-1a to the nuclear speckles [11,12] and has been suggested to couple pre-mRNA splicing with transcription [12]. Acinus was originally identified as a target of caspase-3, a cysteine protease involved in activating chromatin condensation and nuclear fragmentation during apoptosis, yet the role of this protein during normal cellular growth has not been determined [13]. Acinus contains several RS domains and a region similar to the RNA-recognition motif of Drosophila splicing regulator Sxl, and has been suggested that it may be implicated in the regulation of RNA splicing [14–16]. The lamin B receptor (LBR) is the most thoroughly studied protein of the second group of RS domain proteins, i.e. those that do not contain an RRM. LBR is an inner nuclear membrane protein, consisting of a long, hydrophilic N-terminal domain, seven or eight hydrophobic segments that are predicted to span the membrane, and a hydrophilic C-terminal tail [17]. The N-terminal part of the molecule protrudes to the nucleoplasm and contains

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multiple serine-arginine motifs that are phosphorylated by the SRPK1 and the cdc2 kinases [18–20]. LBR is one of the factors that have been implicated in chromatin anchorage to the nuclear envelope. Its N-terminal domain has been reported to bind double-stranded DNA [21], histones H3 and H4 [22] and Hetechromatic Protein 1 (HP1) [23]. A common characteristic of SR proteins and LBR is their tendency to form aggregates. In vitro studies of several SRproteins revealed that the unphosphorylated form of the proteins were insoluble under native conditions [24,25]. Moreover, the fraction of recombinant ASF/SF2 recovered in soluble form is greatly increased in bacteria by coexpression of SRPK1 [26]. LBR was also shown to self-associate through its N-terminal domain and form oligomers that are suggested to bind heterochromatin [27]. In the present study we extend these observations, demonstrating that the RS domains of purified recombinant LBR, PGC-1a and Acinus associate mainly with nuclear RNA and that this interaction is critical in retaining the proteins in a soluble form. We therefore propose that the ability to interact with RNA is a general property of RS domains. Phosphorylation by SRPK1 impedes RNA association, yet it increases the soluble fraction of the RS domain-containing proteins, in a way similar to RNA association. Our findings also point to a global role of SRPK1mediated phosphorylation of the serine residues within an RS domain in switching its ability to interact with RNA and promoting the assembly of dissimilar complexes. 2. Materials and methods 2.1. Construction of plasmids and expression of fusion proteins The pGEX-2T bacterial expression vector (Amersham Pharmacia Biotech) was used to construct plasmids that encode the full-length or various regions of SRPK1, LBR, Acinus and PGC-1 proteins fused with glutathione S-transferase

Fig. 1. LBR, PGC-1 and Acinus expressed in E. coli as GST fusion proteins are associated with bacterial RNA that is indispensable for retaining the fusion proteins in a soluble form. (A) The nucleic acids associated with GST-LBRNt, GST-PGC-1 and GST-Acinus were analyzed in an agarose gel stained with ethidium bromide. Two to three μg of each fusion protein were incubated without or with 30 units DNAse I or 5 μg RNAse for 15 min at 30 °C, prior to agarose gel analysis. A 1-kb DNA ladder is shown on the left. (B) Approximately 2 μg of GST, GST-LBRNt, GST-PGC-1 and GST-Acinus were incubated with 5 μg RNAse for 15 min at 30 °C and the samples were subsequenly centrifuged at 13000 g for 15 min in a microcentrifuge. The supernatants (S) and pellets (P) were analyzed by SDS-PAGE on a 12% (GSTLBRNt) or a 10% gel (GST-PGC-1, GST-Acinus) and then stained with Coomassie Brilliant Blue R-250. Bars on the left indicate molecular masses (in kDa). Only the relevant parts of the Coomassie stained gels are shown.

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(GST). SRPK1 and the N-terminal domain of chicken LBR (LBRNt, amino acids 1-205) were prepared as GST fusion proteins as previously described [18,28]. The cDNAs of LBR(62-205), coding for amino acids 62 to 205, and LBR(92-205), coding for amino acids 92-205, of chicken LBR were amplified with PCR using the cDNA of LBRNt as template. The sense primers were as follows: 5′-CGCGGATCCCAGAGGAAAAGCCAGTCT-3′, LBR(62-205) and 5′-GCGGGATCCAGACGTCGCTCTTCTTCCCATAG-3′, LBR(92-205), while the antisense primer was the same for both constructs: 5′-GCGAATTCTCACTTCCACCAAATTCTAG-3′. The PCR products were purified using the QIAEX® II Gel extraction kit (QIAGEN Inc.), digested with EcoRI and BamHI, repurified, and ligated into the BamHI/EcoRI site of pGEX-2T. Similarly, the cDNA coding for amino acids 767 to 1341 of Acinus L (AF124726, kindly provided by Y. Tsujimoto, Department of Medical Genetics, Osaka University Medical School) was amplified with PCR from the entire Acinus L cDNA with the upstream primer 5′-GCGGGATCCGAGGAGAAGGAGGAAGTGA CCATG-3′ and downstream primer 5′-CCCGGAATTCCTAGCGGCGCCCACCCC GGTCC-3′ and ligated into the BamHI/EcoRI site of pGEX2T. An oligonucleotide-directed in vitro mutagenesis system (Altered Sites RII, In Vitro Mutagenesis System, Promega) was used to delete the two RS domains in the C-terminal region of Acinus L (1175RSRSRSR1181 and 1325SRSRSRS1331 respectively). Using the oligonucleotides 5′-CTTGCGGCGGCGGTCGGGCCCTTCTCGAAC-3′ and 5′-GTCCCGCACAGGT GTGTGGCGCTTGGTGTC-3′ the codons for amino acids 1175 to 1181 and 1325 to 1331 were deleted respectively (ΔRSAcinus). The mutated cDNA was sequenced and subcloned into the pGEX-2T expression vector as described for Acinus L. GST-PGC-1a was a kind gift of M. Hadzopoulou (Laboratory of Developmental Biology, Department of Biology, Aristotle University of Thessaloniki) To delete amino acids 566 to 625 (construct termed GST-ΔRSPGC-1) we first generated the cDNAs coding for amino acids 1 to 565 and 626 to798 respectively of human PGC1a (NM013261). The PCR amplification of the first DNA was performed with the sense primer 5′-GCGGGATCCATGGCGTGGGACATGTGCAAC-3′ and antisense primer 5′-TCCCCCGAGCATCCTTTGGGGTCTTTGAGAAAA-3′, while 5′-TCCCTCGGGCCCTACAGCCGTCGGCCCAGG-3′ was used as sense and 5′-TCCCCCGGGCCTGCGCAAGCTTCTCTGAGCTTC-3′ as antisense primer for the amplification of the second cDNA. The products were digested with BamHI/AvaI and AvaI/SmaI respectively, repurified, and cloned into the BamHI/ SmaI site of pGEX-2T. Escherichia coli strains BL-21 were transformed by standard methods. GST fusion proteins were produced in bacteria and purified using glutathione-Sepharose beads according to the manufacturer's instruction (Amersham Pharmacia). To eliminate the associated nucleic acids, bacterial preparations of GST-LBRNt, GSTPGC-1 and GST-Acinus (2-3 μg of recombinant protein in each case) were treated with 30 units DNAse I (Invitrogen, RNAse free) or 5 μg RNAse (Applichem GmbH, DNAse free) for 15 min at 30 °C. Soluble recombinant proteins depleted from the associated bacterial RNA were prepared by adding 1 M NaCl to all steps of the respective purification procedures, except the last step, where fusion proteins were recovered from the beads by incubation in the normal elution buffer, consisting of 50 mM Tris-HCl pH 8 and 10 mM reduced glutathione. SRPK1 was subcloned into the p-FLAG-CMV-2 vector and expressed in 293T cells with a FLAG tag fused at its N-terminus as previously described [29].

2.2. Kinase assays SRPK1 activity was determined by measuring the incorporation of PO3− 4 from [γ-32P]ATP (6000 Ci/mmol; ICN Pharmaceuticals Ltd) to bacterially expressed GST-LBRNt, GST-Acinus and GST-PGC-1. Routine assays were carried out at 30 °C in a total volume of 25 μl containing 12 mM Hepes pH 7.5, 5-15 mM MgCl2 (as indicated), 50 μM [γ-32P]ATP, 2-3 μg of the appropriate substrate and 0.3 μg GST-SRPK1. Samples were incubated for 30 min at 30 °C and the reaction was stopped by adding 6 μl 5 × SDS sample buffer and heating at 95 °C for 3 min. Incorporation of radioactivity was measured by excising the respective radioactive bands from an SDS–PAGE gel and scintillation counting.

2.3. RNA-binding assays Electrophoretic mobility shift assays, using purified recombinant proteins depleted of bacterial RNA, were performed in RNA binding buffer (20 mM

Hepes pH 7.6, 100 mM KCl, 2 mM EDTA and 0.01% NP-40). The probe was produced by linearizing recombinant pGEM®-T easy vector, that contained a 103 bp DNA insert encoding a fragment of human TERT [for details see ref. [30]], with NotI and then in vitro transcribing the template using T7 RNA polymerase in the presence of radioactively labelled CTP (using a “Riboprobe” kit from Promega). The size of the full-length RNA probe, produced by in vitro transcription, was 165 nucleotides. Samples were analyzed on a 5% polyacrylamide gel in 0.25% TBE. Phosphorylation of GST-LBRNt was achieved by incubating the recombinant protein with GST-SRPK1 (0.3 μg) in kinase assay buffer supplemented with 0.2 mM ATP for 1 h at 30 °C, prior to incubation with the probe. To test whether phosphorylation by SRPK1 could detach the RNA probe from GST-LBRNt, the recombinant protein was first incubated with the radiolabelled probe for 20 min at room temperature and then for another 30 min at 30 °C with GST-SRPK1 in the presence of 0.2 mM ATP. The final

Fig. 2. Bacterial RNA binds to the RS domain of LBR. (A) Schematic diagram of N-terminal fragments of chicken LBR expressed as GST fusion proteins. On top of the figure the amino acid sequence of the RS domain is shown. (B) GSTLBRNt, GST-LBR(62-205) and GST-LBR(92-205) were expressed in E. coli, purified with glutathione-Sepharose, analyzed by SDS-PAGE on a 12% gel and then stained with Coomassie Brilliant Blue R-250 (left panel). Bars on the left indicate molecular masses (in kDa). The asterisks denote the position of fulllength GST-LBRNt, GST-LBR(62-205) and GST-LBR(92-205) respectively. The lower bands represent degradation products (see also Ref. [19]). On the right panel the bacterial RNA associated with GST-LBRNt, GST-LBR(62-205) and GST-LBR(92-205) (2-3 μg of each fusion protein) was analyzed in an agarose gel stained with ethidium bromide. A 1-kb DNA ladder is shown on the left. (C) Approximately 2 μg of GST-LBR(62-205) and GST-LBR(92-205) were incubated with 5 μg RNAse for 15 min at 30 °C and the samples were subsequenly centrifuged at 13000 g for 15 min in a microcentrifuge. The supernatants (S) and pellets (P) were analyzed by SDS-PAGE on a 12% gel and then stained with Coomassie Brilliant Blue R-250. Only the relevant parts of the Coomassie stained gels are shown.

E. Nikolakaki et al. / Biochimica et Biophysica Acta 1780 (2008) 214–225 concentration of magnesium chloride in the phosphorylation reaction was adjusted to 7.5 mM.

2.4. Purification of SR proteins from cell extracts Human 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum and antibiotics. 5 × 105 cells (∼50% confluent) were transfected with 5 μg of p-FLAG-CMV-2 (mock transfection) or p-FLAG-CMV-2-SRPK1 DNA using the calcium phosphate method [31]. Total amounts of plasmid DNA were made up to 20 μg with pcDNA3 (Invitrogen). After 16 h the medium was changed and the cells were incubated for another 24 h. Cells were lysed with 200 μl of 1% Triton buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 μg/ml aprotinin, and 1 mM PMSF) for 30 min on ice and whole cell extracts were clarified by centrifugation at 13000 g for 15 min in a microcentrifuge. Thirty microliters (∼300 μg) of each lysate were transferred to clean tubes, and MgCl2 or RNAse were added to 20 mM or 1 μg / 10 μg lysate respectively. After a 1-h incubation on ice, tubes were centrifuged at 13000 g for 30 min. After careful removal of the supernatants, the pellets were dissolved in sample electrophoresis buffer and analyzed on 10% SDS-PAGE. The identity of the precipitated SR proteins was confirmed by western blotting using the mAb104 monoclonal antibody (culture supernatant, kindly provided by J. Tazi, Institut de Génétique Moléculaire, UMR 5535, C.N.R.S., 34293 Montpellier, France). SRPK1 was detected by Western blotting, using an anti-SRPK1 monoclonal antibody (BD Biosciences Pharmingen, San Jose, CA USA). Following washing with PBST (20 mM phosphate buffer, pH 7.4, 150 mM NaCl, and 0.04% Tween 20), blots were incubated with an alkaline phosphatase-coupled goat anti-mouse secondary antibody, and 5bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate. To determine the associated kinase activity, MgCl2 or RNAse pellets were resuspended in kinase assay buffer supplemented with 50 μM [γ-32P]ATP, and incubated at 30 °C for 30 min. The phosphorylated proteins were analyzed on 10% SDS-PAGE and detected by autoradiography. RNAse pellets were also incubated in kinase assay buffer supplemented with 0.2 mM ATP for 1 h at 30 °C and then analyzed by Western blotting using the mAb04 monoclonal antibody to determine whether the amount of phosphorylated SR proteins has increased after the kinase assay.

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2.5. Disorder predictions Predictions of intrinsic disorder in LBRNt, Acinus and PGC-1 were carried out using a well-characterized disorder predictor PONDR® VL-XT [for pertinent information see ref. 32 and refs. therein]. The cumulative distribution function (CDF) represents a cumulative histogram of the PONDR® VL-XT prediction scores for each residue in a given protein. This histogram allows the separation of ordered and disordered proteins based on the distribution of the disorder scores [33]. The boundary points on the CDF plot were calculated as previously described [34]. The charge-hydropathy (CH) method, initially developed by Uversky et al. [35], was previously used to classify SR proteins as ordered or disordered [32]. The mean net charge and the mean normalized Kyte–Doolittle hydropathy [36] were calculated for each protein and their values were plotted against each other. The boundary between ordered and disordered proteins was determined using a linear discriminant function as previously described [33,37]. Access to PONDR® VL-XT was provided by Molecular Kinetics (Indianapolis, IN). VL-XT is copyright© 1999 by the WSU Research Foundation, all rights reserved. PONDR® is copyright© 2004 by Molecular Kinetics, all rights reserved.

3. Results 3.1. The RS domain of LBR, Acinus and PGC-1a, associates mainly with nuclear RNA that is indispensable for retaining the proteins in a soluble form The N-terminal domain of LBR (LBRNt) has been widely studied in vitro and is readily produced in E. coli. In agreement with recent data demonstrating in vitro oligomerization of LBRNt [27], we noticed that the insoluble fraction of GSTLBRNt was progressively increasing after repeated freezing and thawing. We also noticed that an additional two RS domaincontaining proteins, PGC-1 and Acinus, had the same tendency

Fig. 3. Bacterial RNA binds to the RS domain of GST-PGC-1. (A) Schematic diagram of PGC-1a and ΔRSPGC-1 expressed as GST fusion proteins. The two boxes, marked RS and RRM, represent the RS domain and the RNA Recognition Motif, respectively. On top of the figure the amino acid sequence of the RS domain is shown. (B) GST-PGC-1, and GST-ΔRSPGC-1 were expressed in E. coli, purified with glutathione-Sepharose, analyzed by SDS-PAGE on a 10% gel and then stained with Coomassie Brilliant Blue R-250 (left panel). In the middle panel the bacterial RNA associated with GST-PGC-1 and GST-ΔRSPGC-1 (2-3 μg of each fusion protein) was analyzed in an agarose gel stained with ethidium bromide. A 1-kb DNA ladder is shown on the left. Approximately 1.5 μg of GST-ΔRSPGC-1 were incubated with 5 μg RNAse for 15 min at 30 °C and the samples were subsequenly centrifuged at 13000 g for 15 min in a microcentrifuge. The supernatants (S) and pellets (P) were analyzed by SDS-PAGE on a 12% gel and then stained with Coomassie Brilliant Blue R-250 (right panel). Bars on the left indicate molecular masses (in kDa). Only the relevant part of the Coomassie stained gels is shown.

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to aggregate in the course of time, when produced in bacteria as GST-fusion proteins. Furthermore, the preparations of all three recombinant proteins exhibited a UV absorption ratio, A260/ A280, ranging from 1.1 to 1.4, suggesting that they contained nucleic acids. The association of bacterial nucleic acids to recombinant RS domain-containing proteins was confirmed using horizontal submarine agarose gels (Fig. 1A). To discriminate between DNA and RNA we treated the purified bacterial preparations of GST-LBRNt, GST-PGC-1 and GST-Acinus with 30 units DNAse I (RNAse free) or 5 μg RNAse (DNAse free) for 15 min at 30 °C prior to agarose gel analysis. As shown in Fig. 1A, RNA was present in all bacterial preparations. This was surprising since the isolation of GST-fusion proteins was not handled with any special care. We then asked whether the progressive decrease of the fraction of recombinant proteins recovered in soluble form was a result of RNA hydrolysis. Fig. 1B shows that RNA elimination, following RNAse treatment,

greatly increased the insoluble fraction of recombinant proteins, implying that RNA association was critical for retaining the proteins in a soluble form. To define the RNA-association domain, we expressed in E. coli fusion proteins consisting of GST and two subfragments of the N-terminal domain of LBR, shown in Fig. 2A. In the first one (construct termed GST-LBR(62-205)) we deleted residues 1-61 that contain a tudor domain (for relevant information see http://smart.embl-heidelberg.de), while in the second (construct termed GST-LBR(92-205)) we deleted residues 1-91 that contain both the tudor and the RS domains. The tudor domain is found in many proteins that colocalize mainly with RNA or ssDNA-associated complexes in the nucleus, in the mitochondrial membrane, or at kinetochores. It is not known whether this domain binds directly to RNA and ssDNA, or controls interactions with the nucleoprotein complexes. Fig. 2B shows that RNA association is restricted to residues 62-91 that contain the

Fig. 4. The RS domain of LBR PGC-1 and Acinus binds preferentially to nuclear RNA. (A) Approximately 1 μg of GST-LBRNt, GST-LBR(62-205), GST-LBR(92205) (left panel lanes 2, 3 and 4 respectively), GST-PGC-1 and GST-ΔRSPGC-1 (middle panel, lanes 6 and 7 respectively) and GST-Acinus and GST-ΔRSAcinus (right panel, lanes 9 and 10 respectively) were incubated in the presence of a radioactively labelled RNA probe. (B) Approximately 1 μg of GST-LBRNt (lanes 2-8) was incubated in the presence of a radioactively labelled RNA probe in the absence (lane 2) or in the presence of either unlabelled nuclear RNA (lanes 3-5) or doublestranded DNA with the same sequence as the RNA probe (lanes 6-8), in the amounts indicated (left panel). On the right panel, 1 μg of GST-LBRNt (lanes 2-8) was also incubated with a radioactively labelled RNA probe in the absence (lane 2) or in the presence of either ribosomal RNA (rRNA, lanes 3-5) or tRNA (lanes 6-8), in the amounts indicated. All recombinant proteins used in these assays were depleted of the associated bacterial RNA (see Materials and Methods).

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RS domain of LBR Furthermore, RNA digestion of GST-LBR (62-205) preparations resulted in a significant increase in the insolubility of the recombinant protein, whereas GST-LBR(92205) that was not associated with RNA remained soluble even after RNAse treatment (Fig. 2C). Interestingly, deletion of only the five consecutive RS dipeptides of LBR (75RSRSRSRSRS84) resulted in a decrease but not elimination of the associated RNA (data not shown), suggesting that the RNA-association domain comprises of the RS region and its flanking sequences that are also rich in positively charged residues. Similarly, deletion of residues 565-626 in the PGC-1 molecule, that contain three stretches of RS dipeptides (Fig. 3A), resulted in preparations of the respective recombinant protein (termed GST-ΔRSPGC-1) that did not contain RNA (Fig. 3B, left panel). As expected GST-ΔRSPGC-1 remained soluble, following RNAse treatment (Fig. 3B, right panel) At this point, we need to note the existence of an RNA Recognition Motif (RRM, residues 677-710) in the PGC-1 molecule (10). The fact that deletion of the RS domain abolished binding to bacterial RNA suggests that either RRM binds to specific cellular RNA sequences or else that the interaction of bacterial RNA with RRM is weaker than its association with the RS domain. The RNA-binding activity of the various LBRNt, PGC-1 and Acinus constructs was further studied by electrophoretic mobility shift assays. These assays were performed with radioactively labelled RNA transcribed from recombinant pGEM®-T easy vector, that contained a fragment of human TERT (see Materials and methods). All fusion proteins used in these assays were depleted of the associated bacterial RNA (see Materials and methods). As shown in Fig. 4A, left panel, only LBR recombinant proteins containing the RS domain were associating with the RNA probe (Fig. 4A, left panel, lanes 2 and 3). Inversely, deletion of the RS region (construct GST-LBR(92205)) abolished the RNA-binding activity (Fig. 4A, left panel, lane 4). Similarly, GST-PGC-1 and GST-Acinus were able to bind the radiolabelled RNA probe (Fig. 4A, middle and right panels, lanes 6 and 9 respectively), whereas GST-ΔRSPGC-1 and GST-ΔRSAcinus shifted to a much lesser extent (Fig. 4A, middle and right panels, lanes 7 and 10 respectively). The small mobility shift obtained mainly with GST-ΔRSPGC-1, but also with GST-ΔRSAcinus, is probably due to the sensitivity of the method that allows the detection of the few RNA molecules associated with RRM or other RNA-binding regions in the PGC-1 and Acinus molecules. LBR has been reported to bind DNA, and the binding domain has been also mapped to the RS region [21,38]. Yet, binding of the radioactively labelled RNA resisted competition with even more than 10-fold excess of doublestranded DNA with the same sequence as the RNA probe (Fig. 4B, left panel, lanes 6-8). To test the binding efficiencies of different types of RNA we used nuclear, ribosomal and transfer RNA as competitors in our electrophoretic mobility shift assays. As shown in Fig. 4B, the binding of GST-LBRNt to the RNA probe was efficiently competed with nuclear RNA (left panel, lanes 3-5) and rRNA (right panel lanes 3-5), whereas tRNA was a less efficient competitor (right panel, lanes 6-8). A rather poor competition was also observed with synthetic RNAs, such as Poly I-C (data not shown), suggesting that the doublestranded

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character of an RNA (or DNA) molecule renders its association with RS domains less favourable. Similar results were also observed with GST-Acinus and GST-PGC-1 (data not shown). 3.2. Precipitation of RS domain-containing proteins by magnesium chloride or RNAse tratment The use of magnesium chloride to selectively precipitate a set of proteins was first described by Roth et al. [39]. Zahler et al. [40] improved this protocol such that all mAb104 immunoreactive proteins could be purified from a variety of animal cells and tissues. mAb104 binds a conserved phosphoepitope on the family of SR proteins, consisting of the consecutive RS dipeptides. The observed aggregation was based on the assumption that magnesium mediates ionic cross-linking between phosphoserines on adjacent SR proteins. Yet, magnesium may also mediate aggregation of RS domain-containing proteins through its interaction with the negatively charged phosphate backbone of the associated RNA molecules. To test this hypothesis we added increasing concentrations of magnesium chloride to bacterially produced GST-LBRNt, GST-Acinus and GST-PGC-1. As shown in Fig. 5 concentrations of magnesium ions 7.5 mM and higher progressively increased the insoluble fraction of all three unphosphorylated fusion proteins, with the effect being less profound in the case of GST-PGC-1. Interestingly, we did not observe any precipitation when the recombinant proteins were depleted of the associated bacterial RNA (see Materials and methods).

Fig. 5. Precipitation of GST-LBRNt, GST-Acinus and GST-PGC-1 by magnesium chloride. Approximately 1.5-2 μg of GST-LBRNt, GST-Acinus and GST-PGC-1 were incubated with the indicated concentrations of magnesium chloride for 30 min at 4 °C and the samples were subsequenly centrifuged at 13000 g for 15 min in a microcentrifuge. The same amounts of the three recombinant proteins that were depleted of the associated bacterial RNA were also subjected to the same type of precipitation. In each case the supernatants (S) and pellets (P) were analyzed by SDS-PAGE on a 12% (GST-LBRNt) or a 10% gel (GST-Acinus) and then stained with Coomassie Brilliant Blue R-250. Only the relevant parts of the Coomassie stained gels are shown.

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Pursuing this point further, we tested the opposite hypothesis, whether RNAse treatment of a cell extract could result in the isolation of a set of SR proteins similar to the one previously obtained following magnesium-dependent aggregation [40]. Zahler et al. [40] used two salt precipitations (ammonium sulfate 65-90% and then 20 mM magnesium chloride), while we omitted the first treatment with ammonium sulfate to avoid dissociation of the RNA molecules. To elucidate the role of phosphorylation, mediated by SRPK1, in magnesium or RNAse-dependent aggregation of SR proteins, we performed the precipitations in cell extracts from control 293T cells and 293T cells transfected with pCMV-2 vector encoding FLAG-tagged SRPK1. There was no significent difference in the Coomassie stained proteins following precipitation by magnesium chloride or by RNAse treatment (Fig. 6A). Due to the omission of the ammonium sulfate pre-

cipitation step, the complexity of proteins was rather high. To allow a more direct comparison between magnesium and RNAse precipitation we detected the mAb104 immunoreactive proteins in both pellets. Fig. 6B (lanes 1-4) shows that the intensity of the staining was significantly higher in magnesium pellets. We also observed only a slight increase in the intensity of the bands corresponding to the precipitated proteins from cell extracts expressing FLAG-tagged SRPK1, probably due to endogenous phosphorylation. In a following step, both magnesium and RNAse pellets were incubated in kinase assay buffer supplemented with 50 μM [γ-32P]ATP. The same phosphorylated bands were observed in both preparations, however, the intensity of the radioactively labelled bands in the RNAse pellets was considerably higher (Fig. 6C). This was not due to an increased concentration of co-precipitating SRPK1, since both pellets contained

Fig. 6. Precipitation of SR proteins from 293T cells by magnesium chloride or RNAse tratment. (A) Three hundred micrograms of lysates from 293T cells transfected with the pCMV-2 vector alone or with pCMV-2 vectors encoding FLAG-tagged SRPK1 were treated with magnesium chloride or RNAse as described under Experimental Procedures. The precipitated proteins were dissolved in sample electrophoresis buffer, analyzed on 12% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue R-250. (B) An immunoblot of the same proteins stained with mAb104 (lanes 1-4). In lanes 5 and 6 the RNAse pellets, from control and 293T cells overexpressing FLAG-SRPK1 respectively, were resuspended in kinase assay buffer, incubated in the presence of 0.2 mM ATP at 30 °C for 60 min and then subjected to Western blotting analysis with mAb104. (C) The magnesium or RNAse precipitated proteins were incubated in kinase assay buffer supplemented with 50 μM [γ-32P]ATP, at 30 °C for 30 min. Labelled proteins were then analyzed on 12% SDS-PAGE and detected by autoradiography. (D) An immunoblot of the precipitated proteins stained with an anti-SRPK1 monoclonal antibody. Bars on the left indicate molecular masses (in kDa).

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comparable amounts of associated SRPK1 (Fig. 6D). To address whether the unphosphorylated or partially phosphorylated proteins obtained following RNAse treatment are indeed SR proteins, the RNAse pellets were first incubated for 60 min at 30 °C in the presence of cold ATP and then subjected to Western blotting analysis with mAb104. As shown in Fig. 6B (compare lanes 3,4 with lanes 5,6) the amount of mAb104 immunoreactive proteins has noticeably increased after the kinase assay. From the sum of these observations it can be concluded that magnesium chloride mediates the aggregation mainly of phosphorylated SR proteins, whereas RNAse treatment of the cell extracts results in the precipitation of RNA-associated but unphosphorylated or partially phosphorylated SR proteins. 3.3. RNA association inhibits phosphorylation by SRPK1, while SRPK1-mediated phosphorylation prevents RNA binding to the RS domain Given that unphosphorylated or partially phosphorylated RS domain-containing proteins seem to associate with RNA molecules we then tested whether RNA association prevents phosphorylation by SRPK1. To this end, GST-LBRNt, GSTAcinus and GST-PGC-1 were used as substrates for in vitro phosphorylation assays with SRPK1. Results presented in Fig. 7A reveal that RNA elimination, following RNAse treatment, resulted in an average 2-fold increase in the phosphorylation efficiency of the recombinant proteins. A close correlation was also noted between the increase in phosphorylation efficiency and magnesium concentration. As shown in Fig. 7B, the

Fig. 7. RNA association inhibits phosphorylation by SRPK1, while SRPK1mediated phosphorylation prevents RNA binding to GST-LBRNt and retains the fusion protein in a soluble form. (A) Approximately 2 μg of GST-LBRNt, GSTPGC-1 and GST-Acinus were incubated without or with 5 μg RNAse for 15 min at 30 °C and then in vitro phosphorylated by GST-SRPK1 in kinase assay buffer supplemented with 50 μM [γ-32P]ATP. The samples were analyzed by SDSPAGE and then stained with Coomassie Brilliant Blue R-250 and autoradiographed. Only the relevant parts of the Coomassie stained (left part) and autoradiographed gels (right part) are shown. In the panels showing LBRNt protein the upper band corresponds to full-length GST-LBRNt, while the lower bands represent degradation products (see also Fig. 2 and Ref. [19]). (B) Approximately 2 μg of GST-LBRNt were incubated without or with 5 μg RNAse for 15 min at 30 °C and then in vitro phosphorylated by GST-SRPK1, in the presence of the indicated magnesium concentrations. Only the relevant part of the autoradiographed gel is shown. Both in A and B, incorporation of radioactivity was measured by excising the respective radioactive bands and scintillation counting. (C) left panel: Approximately 1 μg of GST-LBRNt (depleted of bacterial RNA) was preincubated without (lane 2) or with GSTSRPK1 (lane 3) in kinase assay buffer supplemented with 0.2 mM ATP for 1 h at 30 °C and then incubated in the presence of a radioactively labelled RNA probe; right panel: Approximately 2 μg of GST-LBRNt were incubated with 5 μg RNAse or with 5 μg RNAse and GST-SRPK1 in kinase assay buffer supplemented with 0.2 mM ATP for 1 h at 30 °C. The samples were subsequenly centrifuged at 13000 g for 15 min in a microcentrifuge. The supernatants (S) and pellets (P) were analyzed by SDS-PAGE on a 12% gel and then stained with Coomassie Brilliant Blue R-250. Only the relevant part of the Coomassie stained gel is shown. (D) GST-LBRNt (∼1 μg, depleted of bacterial RNA) was incubated with the radiolabelled RNA probe for 20 min at room temperature (lane 2) or with the radiolabelled RNA probe for 20 min at room temperature and then in kinase assay buffer containing 0.2 mM ATP for another 30 min at 30 °C, either in the absence (lane 3) or in the presence of GST-SRPK1 (lane 4).

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lower the magnesium concentration, the less efficient the phosphorylating activity of SRPK1 and the higher its dependence on RNA elimination. When kinase assays were performed in the presence of 5 mM MgCl2 RNAse treatment resulted in a ∼4 fold increase in the extent of phosphorylation by SRPK1. No such increase was observed at 15 mM MgCl2, implying that part of the magnesium concentration was not required for the phosphorylation reaction itself, but was rather used for the interaction with the RNA molecules that are associated with the substrate. Consistent with the data described above, phosphorylation of the RS domain of LBRNt by SRPK1 impeded its interaction with RNA (Fig. 7C, left panel). Furthermore, phosphorylation greatly increased the fraction of GST-LBRNt recovered in soluble form, thereby mimicking the RNA effect (Fig. 7C, right panel). Continuing these studies, we then examined whether phosphorylation could dissociate the RNA molecules from the RS domain. In this respect, GST-LBRNt was first incubated with the radiolabelled RNA probe for 20 min at room temperature and then for another 30 min at 30 °C with GST-SRPK1 in the presence of cold ATP. As shown in Fig. 7D phosphorylation by SRPK1 not only prevented RNA binding but also was able to

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detach the associated RNA molecules. From these observations we conclude that SRPK1-mediated phosphorylation of the serine residues within an RS domain switch its ability to interact with RNA, and may therefore promote the assembly of dissimilar complexes. 4. Discussion 4.1. A model depicting the aggregated and soluble states of RS domain-containing proteins A model accommodating all of our observations is presented in Fig. 8. RS domain-containing proteins may occur in two different states: the aggregated state when they are unmodified and the soluble state when they are associated with RNA molecules or when the serine residues within the RS domain are phosphorylated. Soluble proteins can switch from an RNA-associated to a phosphorylated form and vice-versa by a phosphorylation or, respectively, by a dephosphorylation reaction (mediated most likely by specific protein phosphatases) coupled with RNA association. Dissociation of RNA and the subsequent oligomerization of soluble proteins may occur exclusively through a phosphorylation/dephosphorylation pathway or through a specific molecular mechanism that mediates the detachment and/or cleavage of the RNA molecules leading directly to the oligomerization of RS domain-containing proteins. 4.2. Aggregation of RS domain-containing proteins may be due to their disordered structure At present we can not provide a definitive explanation about the chemical forces that drive the proteins to precipitate or to

become soluble. It is possible that the high positive net charge of the RS domain(s) due to the presence of multiple consecutive arginines makes RS domain-containing proteins more susceptible to self-association and aggregation, while alternating negative and positive charges upon RNA binding or phosphorylation decrease their mean net charge and render them to a more soluble state. It should be noted that a high mean net charge (together with a low mean hydrophobicity) is a distinctive characteristic of a novel recently discovered class of proteins, e.g. intrinsically disordered proteins [33,37]. Incidentally, SR proteins were found to have properties characteristic of such intrinsically disordered proteins and probably lack a folded structure under physiological conditions [32]. Their RS domains, in particular, were predicted to be completely unstructured [32]. Following these observations we performed extensive bionformatics analysis of LBRNt, Acinus (767-1341) and PGC-1 using the disorder predictor PONDR® VL-XT (Fig. 9) as well as cumulative distribution function (Fig. 10A) and charge-hydrophathy analysis (Fig. 10B) (see Experimental Procedures). According to our predictions, all three proteins are highly disordered with the overall percent disorder of 75.8%, 69.3% and 55.8% for Acinus, LBRNt and PGC-1, respectively. In agreement with previous observations [32], the RS domains of all three proteins are predicted to be disordered, whereas the RRM domain of PGC-1 is predicted to be ordered (Fig. 9). Interestingly, the tudor domain of LBRNt is also predicted to be ordered. The CDF and CH analysis agree with each other as well as with PONDR® VL-XT per-residue predictions; they both classify all three proteins as members of the disordered protein family (Fig. 10A and B). The analysis of Acinus, LBRNt and PGC-1 performed here together with the previous analysis of SR splicing factors [32] suggest that intrinsic structural disorder may be a general feature of all RS domain-

Fig. 8. A provisional model for the oligomerization state of RS domain-containing proteins. Unmodified RS domain-containing proteins are found in an aggregated form. Association with RNA molecules or phosphorylation of the serine residues within the RS domain renders the proteins soluble. Phosphorylation may be mediated solely by members of the SRPK family or else by members of both the SRPK and CLK family. CLKs for instance efficiently phosphorylate SR proteins, whereas they are unable to phosphorylate the RS domain of LBR [for pertinent information see ref. [52]]. Soluble proteins can switch from an RNA-associated to a phosphorylated form and vice-versa by a phosphorylation or, respectively, by a dephosphorylation reaction coupled with RNA association. Oligomerization of soluble proteins may occur through dephosphorylation or through the detachment and/or cleavage of the RNA molecules.

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Fig. 9. Disorder predictions for chicken LBRNt (aa 1-205), human PGC-1 and human Acinus L C-terminus (aa 767-1341). The upper scale numbering corresponds to LBR and PGC-1, whereas the bottom scale numbering corresponds to Acinus L. The thick upper bar for each protein represents PONDR® VL-XT predictions with the red color signifying disorder and the blue color signifying order (see the gradient representation of the prediction scores shown on the vertical bar of the legend). The lower black bar represents the location of the RS domains, the lower yellow bar represents the location of the RRM domain of PGC-1.

containing proteins, and that high disorder content may be a key reason rendering them prone to aggregation. 4.3. The role of RNA and phosphorylation on the assembly of macromolecular complexes by LBR at the nuclear periphery Intrinsically disordered proteins are often involved in interactions with multiple partners and the assembly of macromolecular structures due to their conformational flexibility and adaptability [41,42]. Such macromolecular complexes are often assembled by several integral proteins of the nuclear envelope (e.g. LAP1, LAP2, and LBR) that are proposed to function as chromatin-anchorage or chromatin-remodeling platforms [for review see ref. [43]. Previous data [27], as well as data from this work clearly demonstrate that LBR self-associates through its N-terminal domain and forms oligomers. Interestingly, the native protein was also shown to form microdomains at the level of the nuclear envelope [27]. The current view is that LBR binds heterochromatin as a higher oligomer. Challenging this view, we propose that the N-terminal domains of several adjacent LBR molecules exhibit an “anemone” structure at the inner nuclear membrane. More specifically, when their RS domains are unphosphorylated or they are not associated with RNA (or DNA, see below) they self-associate and exhibit a closed configuration that is inactive and does not bind chromatin. Conversely, phosphorylation greatly increases the solubility of the RS domains, thereby transforming them into protruding segments into the nucleoplasm that mediate chromatin anchorage. In line with this hypothesis, Takano et al. [20] demonstrated that SRPK1-mediated phosphorylation significantly stimulates chromatin association. The important question is whether RNA is found associated with the N-terminal domain of LBR and if so what type of RNA. Alternatively, due to the high DNA concentration of peripheral heterochromatin, DNA may function in associating with and opening the closed configuration of LBRNt, thereby facilitating the binding of histones. LBRNt showed a clear preference for RNA association rather than DNA. In addition, nuclear RNA was more efficient in competition experiments than tRNA, and to a lesser extent than rRNA, suggesting some degree of specificity in the binding. It must be noted that other RNA-

Fig. 10. Classification of chicken LBRNt, human PGC-1 and human Acinus L Cterminus using the CDF and CH analysis. (A) CDF analysis of chicken LBRNt, human PGC-1, and human Acinus L C-terminus. CDF curves were constructed from PONDR® VLXT prediction scores as described in Experimental Procedures. The order-disorder boundary is represented by the solid line with open circles. CDF curves for disordered proteins are located below the boundary. (B) Charge-Hydropathy (CH) analysis of chicken LBRNt, human PGC-1, and human Acinus L C-terminus. Mean net charge and mean hydropathy for each protein were calculated as described in Experimental Procedures. The ordered proteins are represented as solid circles, the disordered proteins as open circles and LBRNt, PGC-1 and Acinus L as grey square, triangle and diamond, respectively. The order-disorder boundary is shown as a solid line.

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associated proteins, such as the Drosophila chromodomain proteins MOF and MSL-3, show no or little binding specificity in vitro, although their physiological target is roX RNA [44]. An intriguing possibility is that RNA association or phosphorylation may promote different interactions. HP1 has been reported to interact with LBRNt [23]. It was also shown that the association of HP1 with pericentromeric heterochromatin is mediated by an RNA binding activity present in the hinge region of the protein [45]. It is therefore possible that LBRNt associated with RNA acts as a docking site for HP1 at the nuclear envelope. It should be noted that HP1 was found temporarily associated with the nuclear envelope before incorporation into specific areas in the nucleus [46]. Conversely, phosphorylated LBRNt may act solely as a chromatin anchorage site. 4.4. SRPKs are linked with the production of small noncoding RNAs More than twenty years ago, electron microscopy studies suggested that some species of snRNA might be involved in the structure of chromatin [47]. In Chinese Hamster Ovary (CHO) cells several small nuclear RNAs (snRNAs) were found in highest density in the peripheral part of the nucleus, especially over areas of condensed chromatin. Furthermore, transcripts originating from the intergenic spacer that separates rRNA genes (rDNA) are required for establishing and maintaining a specific heterochromatic configuration at the promoter of a subset of rDNA arrays [48]. Interestingly, a direct link between SRPKs and small non-coding RNAs was recently established by the identification of SRPK1a, which is an alternatively spliced form of SRPK1 [29], as a component of the microprocessor complex that mediates the genesis of microRNAs (miRNAs) [49]; see also Biomolecular Interaction Network Database]. MiRNAs are a growing family of non-protein coding RNAs that regulate the expression of homologous target-gene transcripts and maybe the high-order chromatin structure. About a quarter of human miRNA genes are located in introns of pre-mRNAs and since they have the same orientation as pre-mRNAs, it is likely that they are processed from the introns. In this respect, it should be noted that the RNase III endonuclease Drosha that is the catalytic machinery of the microprocessor complex and SRPK1 have also been shown to participate in pre-ribosomal RNA processing [50,51]. Collectively our data support the concept that the oligomerization state of RS domain-containing proteins may promote efficient and coordinated cellular processes through the assembly or disassembly of specific higher-order macromolecular structures. Deciphering the phosphorylation events that take place in the microenvironment of each individual RS domain together with the identification and characterization of the associated RNA molecules will ultimately contribute to an understanding of the functions of RS domain-containing proteins. Acknowledgments We thank Y. Tsujimoto for providing us with the Acinus L cDNA clone, M. Hadjopoulou for the GST-PGC-1a construct, J.

Tazi for the mAb104 monoclonal antibody, and A. Tsiftsoglou for providing us with a tissue culture facility. We also thank S.D. Georgatos and J.G. Georgatsos for valuable discussions. and comments on the manuscript. This work was supported in part by grants from the Greek Ministry of Education (HRAKLEITOS No 21768 and PYTHAGORAS II No 80913) to T.G. and by the U.S. National Science Foundation grant #MCB 0444818 to L.M.I. Ioannis Sanidas and Philippos Peidis were recipients of a fellowship from the Greek State Scholarship's Foundation (I.K.Y.). References [1] H. Li, P.M. Bingham, Arginine/serine-rich domains of the su(wa) and tra RNA processing regulators target proteins to a subnuclear compartment implicated in splicing, Cell 67 (1991) 335–342. [2] R. Boucher, C.A. Ouzounis, A.J. Enright, B.J. Blencowe, A genome-wide survey of RS domain proteins, RNA 7 (2001) 1693–1701. [3] B.R. Graveley, T. Maniatis, Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing, Mol. Cell 1 (1998) 765–771. [4] J.E. Mermoud, P.T. Cohen, A.I. Lamond, Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism, EMBO J. 13 (1994) 5679–5688. [5] B.R. Graveley, Sorting out the complexity of SR protein functions, RNA 6 (2000) 1197–1211. [6] J. Valcarcel, R.K. Gaur, R. Singh, M.R. Green, Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA, Science 273 (1996) 1706–1709. [7] H. Shen, J.L. Kan, M.R. Green, Arginine-serine-rich domains bound at splicing enhancers contact the branchpoint to promote prespliceosome assembly, Mol. Cell 13 (2004) 367–376. [8] H. Shen, M.R. Green, A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly, Mol. Cell 16 (2004) 363–373. [9] H. Shen, M.R. Green, RS domains contact splicing signals and promote splicing by a common mechanism in yeast through humans, Genes Dev. 20 (2006) 1755–1765. [10] J. Lin, C. Handschin, B.M. Spiegelman, Metabolic control through the PGC-1 family of transcription coactivators, Cell Metab. 1 (2005) 361–370. [11] T. Shiraki, N. Sakai, E. Kanaya, H. Jingami, Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state, J. Biol. Chem. 278 (2003) 11344–11350. [12] M. Monsalve, Z. Wu, G. Adelmant, P. Puigserver, M. Fan, B.M. Spiegelman, Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1, Mol. Cell 6 (2000) 307–316. [13] S. Sahara, M. Aoto, Y. Eguchi, N. Imamoto, Y. Yoneda, Y. Tsujimoto, Acinus is a caspase-3 activated protein required for apoptotic chromatin condensation, Nature 401 (1999) 168–173. [14] J. Rappsilber, U. Ryder, A.I. Lamond, M. Mann, Large-scale proteomic analysis of the human spliceosome, Genome Res. 12 (2002) 1231–1245. [15] Z. Zhou, L.J. Licklider, S.P. Gygi, R. Reed, Comprehensive proteomic analysis of the human spliceosome, Nature 419 (2002) 182–185. [16] C. Schwerk, J. Prasad, K. Degenhardt, H. Erdjument-Bromage, E. White, P. Tempst, V.J. Kidd, J.L. Manley, J.M. Lahti, D. Reinberg, ASAP, a novel protein complex involved in RNA processing and apoptosis, Mol. Cell. Biol 23 (2003) 2981–2990. [17] H.J. Worman, C.D. Evans, G. Blobel, The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains, J Cell. Biol. 111 (1990) 1535–1542. [18] E. Nikolakaki, G. Simos, S.D. Georgatos, T. Giannakouros, A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins, J. Biol. Chem. 271 (1996) 8365–8372.

E. Nikolakaki et al. / Biochimica et Biophysica Acta 1780 (2008) 214–225 [19] E. Nikolakaki, J. Meier, G. Simos, S.D. Georgatos, T. Giannakouros, Mitotic phosphorylation of the lamin B receptor by a serine/arginine kinase and p34(cdc2), J. Biol. Chem. 272 (1997) 6208–6213. [20] M. Takano, Y. Koyama, H. Ito, S. Hoshino, H. Onogi, M. Hagiwara, K. Furukawa, T. Horigome, Regulation of binding of lamin B receptor to chromatin by SR protein kinase and cdc2 kinase in Xenopus egg extracts, J. Biol. Chem. 279 (2004) 13265–13271. [21] Q. Ye, H.J. Worman, Primary structure analysis and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane, J. Biol. Chem. 269 (1994) 11306–11311. [22] H. Polioudaki, N. Kourmouli, V. Drosou, A. Bakou, P.A. Theodoropoulos, P.B. Singh, T. Giannakouros, S.D. Georgatos, Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1, EMBO Rep. 2 (2001) 920–925. [23] Q. Ye, H.J. Worman, Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1, J. Biol. Chem. 271 (1996) 14653–14656. [24] H. Ge, P. Zuo, J.L. Manley, Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators, Cell 66 (1991) 373–382. [25] A.R. Krainer, A. Mayeda, D. Kozak, G. Binns, Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators, Cell 66 (1991) 383–394. [26] B.G. Yue, P. Ajuh, G. Akusjarvi, A.I. Lamond, J.P. Kreivi, Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli, Nucl. Acids Res. 28 (2000) E14. [27] D. Makatsori, N. Kourmouli, H. Polioudaki, L.D. Shultz, K. McLean, P.A. Theodoropoulos, P.B. Singh, S.D. Georgatos, The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope, J. Biol. Chem. 279 (2004) 25567–25573. [28] S. Papoutsopoulou, E. Nikolakaki, T. Giannakouros, SRPK1 and LBR protein kinases show identical substrate specificities, Biochem. Biophys. Res. Commun. 255 (1999) 602–607. [29] E. Nikolakaki, R. Kohen, A.M. Hartmann, S. Stamm, E. Georgatsou, T. Giannakouros, Cloning and characterization of an alternatively spliced form of SR protein kinase 1 that interacts specifically with scaffold attachment factor-B, J. Biol. Chem. 276 (2001) 40175–40182. [30] V. Kotoula, S. Barbanis, E. Nikolakaki, D. Koufoyannis, C.S. Papadimitriou, G. Karkavelas, Relative expression of human telomerase catalytic subunit (hTERT) transcripts in astrocytic gliomas, Acta Neuropathol. (Berl) 107 (2004) 443–451. [31] C. Chen, H. Okayama, High-efficiency transformation of mammalian cells by plasmid DNA, Mol. Cell. Biol. 7 (1987) 2745–2752. [32] C. Haynes, L.M. Iakoucheva, Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins, Nucl. Acids Res. 34 (2006) 305–312. [33] A.K. Dunker, Z. Obradovic, P. Romero, E.C. Garner, J. Brown, Intrinsic protein disorder in complete genomes, Genome Inform. Ser. Workshop Genome Inform. 11 (2000) 161–171. [34] C.J. Oldfield, Y. Cheng, M.S. Cortese, C.J. Brown, V.N. Uversky, A.K. Dunker, Comparing and combining predictors of mostly disordered proteins, Biochemistry 44 (2005) 1989–2000. [35] V.N. Uversky, J.R. Gillespie, A.L. Fink, Why are ‘natively unfolded’ proteins unstructured under physiologic conditions? Proteins 41 (2000) 415–427.

225

[36] J. Kyte, R.F. Doolittle, A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157 (1982) 105–132. [37] A.K. Dunker, J.D. Lawson, C.J. Brown, R.M. Williams, P. Romero, J.S. Oh, C.J. Oldfield, A.M. Campen, C.M. Ratliff, K.W. Hipps, J. Ausio, M.S. Nissen, R. Reeves, C. Kang, C.R. Kissinger, R.W. Bailey, M.D. Griswold, W. Chiu, E.C. Garner, Z. Obradovic, Intrinsically disordered protein, J. Mol. Graph. Model 19 (2001) 26–59. [38] M. Takano, M. Takeuchi, H. Ito, K. Furukawa, K. Sugimoto, S. Omata, T. Horigome, The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region, Eur. J. Biochem. 269 (2002) 943–953. [39] M.B. Roth, A.M. Zahler, J.A. Stolk, A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription, J. Cell Biol. 115 (1991) 587–596. [40] A.M. Zahler, W.S. Lane, J.A. Stolk, M.B. Roth, SR proteins: a conserved family of pre-mRNA splicng factors, Genes Dev. 6 (1992) 837–847. [41] C. Haynes, C.J. Oldfield, F. Ji, N. Klitgord, M.E. Cusick, P. Radivojac, V.N. Uversky, M. Vidal, L.M. Iakoucheva, Intrinsic disorder is a common feature of hub proteins from four eukaryotic interactomes, PLoS Comput. Biol. 2 (2006) e100. [42] K. Namba, Roles of partly unfolded conformations in macromolecular self-assembly, Genes Cells 6 (2001) 1–12. [43] S.D. Georgatos, The inner nuclear membrane: simple or very complex? EMBO J. 20 (2001) 2989–2994. [44] A. Akhtar, D. Zink, P.B. Becker, Chromodomains are protein-RNA interaction modules, Nature 407 (2000) 405–409. [45] C. Muchardt, M. Guilleme, J.S. Seeler, D. Trouche, A. Dejean, M. Yaniv, Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha, EMBO Rep. 3 (2002) 975–981. [46] N. Kourmouli, P.A. Theodoropoulos, G. Dialynas, A. Bakou, A.S. Politou, I.G. Cowell, P.B. Singh, S.D. Georgatos, Dynamic associations of heterochromatin protein 1 with the nuclear envelope, EMBO J. 19 (2000) 6558–6568. [47] G. Lacoste-Royal, R. Simard, Localization of small nuclear RNA by EM autoradiography in Chinese hamster ovary (CHO) cells, Exp. Cell. Res. 149 (1983) 311–323. [48] C. Mayer, K.M. Schmitz, J. Li, I. Grummt, R. Santoro, Intergenic transcripts regulate the epigenetic state of rRNA genes, Mol. Cell 22 (2006) 351–361. [49] R.I. Gregory, K.P. Yan, G. Amuthan, T. Chendrimada, B. Doratotaj, N. Cooch, R. Shiekhattar, The Microprocessor complex mediates the genesis of microRNAs, Nature 432 (2004) 235–240. [50] H. Wu, H. Xu, L.J. Miraglia, S.T. Crooke, Human RNase III is a 160-kDa protein involved in preribosomal RNA processing, J. Biol. Chem. 275 (2000) 36957–36965. [51] T. Hayano, M. Yanagida, Y. Yamauchi, T. Shinkawa, T. Isobe, N. Takahashi, Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome, J. Biol. Chem. 278 (2003) 34309–34319. [52] E. Nikolakaki, C. Du, J. Lai, T. Giannakouros, L. Cantley, L. Rabinow, Phosphorylation by LAMMER protein kinases: determination of a consensus site, identification of in vitro substrates, and implications for substrate preferences, Biochemistry 41 (2002) 2055–2066.