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Keywords:

  • BC1 RNA;
  • Brain;
  • Neuronal dendrite;
  • Pur α and pur β proteins;
  • Microtubule-binding protein;
  • RNA transport

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

Abstract: Neural BC1 RNA is distributed in neuronal dendrites as RNA—protein complexes (BC1 RNPs) containing Translin. In this study, we demonstrated that the single-stranded DNA- and RNA-binding protein pur α and its isoform, pur β, which have been implicated in control of DNA replication and transcription, linked BC1 RNA to microtubules (MTs). The binding site was within the 5′ proximal region of BC1 RNA containing putative dendrite-targeting RNA motifs rich in G and U residues, suggesting that in the cytoplasm of neurons, these nuclear factors are involved in the BC1 RNA transport along dendritic MTs. The pur proteins were not components of BC1 RNP but appeared to associate with MTs in brain cells. Therefore, it is suggested that they may transiently interact with the RNP during transport. In this respect, the interaction of pur proteins with BC1 RNA could be regulated by the Translin present within the RNP, because the binding mode of these two classes of proteins (pur proteins and Translin) to the dendrite-targeting RNA motifs was mutually exclusive. As the motifs are well conserved in microtubule-associated protein 2a/b mRNA as well, the pur proteins may also play a role(s) in the dendritic transport of a subset of mRNAs.

Neural BC1 RNA is expressed selectively in rodent brain (Sutcliffe et al., 1982; Anzai et al., 1986) and is distributed in neuronal dendrites (Tiedge et al., 1991) in the form of ribonucleoprotein particles (RNPs) (Kobayashi et al., 1991; Cheng et al., 1996). In a previous study, we demonstrated the presence of two copies of y-, h-element homologous sequences in BC1 RNA [nucleotides (nt) 1-22 and 47-76] (see Fig. 1) (Muramatsu et al., 1998), and we detected two RNA-binding proteins using the radiolabeled 3′ distal y-, h-elements (nt 47-76) as a probe, which co-purified with BC1 RNP (Kobayashi et al., 1998; Muramatsu et al., 1998). One of these proteins was mouse Translin and the other was a 37-kDa protein. In addition to these observations, we demonstrated the dendritic distribution of Translin protein in hippocampal neurons in primary culture (Kobayashi et al., 1998), suggesting that Translin is involved in the dendritic translocation of BC1 RNP. Recently, Translin was also detected in dendrites of a subset of neurons of the mouse brain (Wu et al., 1999).

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Figure 1. Nucleotide sequence elements in BC1 RNA relevant to the dendritic translocation. a: The location of y-, h-element homologous sequences and G-, U-rich motifs I-III is indicated by a thin line; the numbers in parentheses indicate the distances, in nucleotide numbers, from the 5′ end of BC1 RNA (Kobayashi et al., 1998). A- and B-boxes, intragenic promoter sequences for RNA polymerase III. b: Nucleotide sequences of motifs I-III and the putative dendrite-targeting motifs present in MAP2a/b mRNA, which are homologous with the G-, U-rich motifs, are shown. c: Subfragments of BC1 RNA used as the probes in EGMSA.

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Wu et al. (1997) reported that testis/brain RNA-binding protein (TB-RBP), which was originally identified as a y-, h-element-binding protein (Kwon and Hecht, 1991), is the mouse homologue of Translin. They also reported that TB-RBP has translational repressor activity (Kwon and Hecht, 1993) and links a subset of mRNAs to microtubules (MTs) in vitro through binding to their y-, h-sequence elements (Han et al., 1995a). These observations in turn suggested that dendritic MTs are also involved in the process of BC1 RNP transport and that this RNP may have regulatory roles in local translation of other dendritic mRNAs through repression of translation. The possible involvement of BC1 RNP in local protein synthesis was also suggested by Kremerskothen et al. (1998), who recently demonstrated the presence of autoantigen La/SS-B as a BC1/BC200 RNA-binding protein, which may provide a link between the 3′ end tail of BC1 RNA and the ribosome. These observations suggest that BC1 RNA is a molecular scaffold for the assembly of BC1 RNP that is required for the dendritic delivery of several protein factors capable of regulating translation within dendrites.

Muslimov et al. (1997) suggested that the dendritetargeting signal of BC1 RNA is contained within a 5′ region that contains no more than 62 nucleotides. Because similar sequence motifs are also present in microtubule-associated protein 2 (MAP2) a/b mRNAs, but not in nondendritic MAP2c mRNA, they are suggested to be cis elements common to both BC1 RNA and a subset of dendritic mRNAs and may direct the dendritic delivery of these RNAs with their corresponding trans-acting factors. However, no information about the proteins has been reported except Translin and the 37-kDa protein. When these putative dendrite-targeting motifs were compared with the two y-, h-elements in BC1 RNA, a short stretch of nucleotide sequences (∼ 14 nt) rich in G and U residues was detected (motifs I-III; see Fig. 1) (Kobayashi et al., 1998), suggesting that they may constitute the core binding sites. We have observed also that the 5′ proximal segment of BC1 RNA (nt 1-34) containing the putative dendrite-targeting motifs (nt 1-12 and 24-34) (Muslimov et al., 1997), which overlap the 5′ proximal y-, h-elements (nt 1-22), interacted with a distinct RNA-binding protein (C2 protein) in addition to Translin protein (unpublished observations). As another y-, h-element (nt 47-76) that is present in BC1 RNA interacted with Translin but not with the C2 protein, it would be interesting to characterize this “dendrite-targeting motif-specific” RNA-binding protein, which may play a role(s), together with Translin, in the process of BC1 RNA transport to the neuronal dendrites.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

Chemicals

Chemicals were purchased from the following companies: T7-MEGAshortscript, Ambion; colcemid and paclitaxel (Taxol), Sigma; poly(A), poly(U), and poly(G), Amersham Pharmacia Biotech; Dynabeads M-280 Streptavidin, Dynal; ProtBlot Western Blot AP Systems, Promega; Immobilon P membranes, Millipore; ProBlott membranes, Perkin-Elmer; GTP and lysylendopeptidase AP-1, Wako Pure Chemicals; oligonucleotides, Espec-Oligo, Japan; and 32P-labeled ribonucleotides, ICN Biomedicals. All other chemicals were of reagent grade.

Preparation of RNA probes

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

32P-BC1 1-4 probes were synthesized by in vitro transcription of the required oligonucleotide templates, containing T7 promoter sequences in the presence of [α-32P]CTP (111 TBq/mmol), and transcription was performed essentially according to the supplier of T7-MEGAshortscript. All the synthesized RNA probes were extracted with phenol/chloroform and then electrophoretically purified on a polyacrylamide gel containing 4 M urea.

Electrophoretic gel mobility shift assays (EGMSAs)

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

EGMSAs were performed essentially according to the method of Han et al. (1995a). For the supershift analysis using anti-pur α or anti-pur β antibodies, extracts of MTs with CaCl2 (see below) were preincubated with 1 μl (500 ng) of anti-pur antibody or control rabbit antibody (500 ng) in the assay mixture containing all the components except the required 32P-labeled probes. After incubation overnight on ice, the 32P-labeled probe was added, and the resulting complexes were analyzed.

Preparation of extracts from MTs reassembled in vitro

Mouse brains were homogenized in piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer [100 mM PIPES (pH 6.9), 1 mM MgCl2, 10 mM EGTA, 10 μg leupeptin/ml, 2 μg pepstatin/ml, and 2 mM phenylmethylsulfonyl fluoride containing 30% (vol/vol) glycerol]. After centrifugation at 15,000 g for 15 min, the supernatant was centrifuged at 200,000 g at 2°C for 30 min. The aliquots of S200 obtained were incubated in 1 mM GTP, 1 mM GTP + 20 μM Taxol, or 1 mM GTP + 20 μM Taxol + 1 mM colcemid at 30°C for 15 min and on ice for 15 min. After centrifugation at 200,000 g at 25°C for 30 min, the MT pellets obtained were dissolved in a CaCl2 solution (100 mM PIPES, pH 6.9, 2 mM CaCl2, 1 mM MgSO4) made to the original volume of the S200 fraction. Insoluble materials were removed by centrifugation at 10,000 g for 5 min, and the supernatant was used as an MT extract. Aliquots of the extracts were diluted appropriately with the CaCl2 solution and analyzed in EGMSA to determine the amounts of C2 protein using excess amounts of 32P-BC1-1 RNA probes.

Preparation of RNA affinity beads

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

BC1-1 RNA with a 25-nt extension at the 3′ end was synthesized by in vitro transcription of the required oligonucleotide template containing T7 promoter sequences according to the instructions of the supplier of T7-MEGAshortscript. A trace amount of [α-32P]CTP was incorporated into the transcript to allow quantitation of RNA. The nucleotide sequence of the template is 5′-TAATACGACTCACTATAGGGGTTGGGGATTTAGCTCAGTGGTAGAGCGCTTGTAGGTCATCAGGCATCGACTAACG-3′. The region corresponding to BC1-1 RNA is underlined. Forty micrograms of the RNA transcript obtained was then annealed with equimolar single-stranded DNA complementary to the 3′ extension of the RNA (5′-CGTTAGTCGATGCCTGATGACCTAC-3′). The 5′ end of this single-stranded DNA was biotinylated. The resulting RNA/DNA duplexes were coupled to Dynabeads M-280 Streptavidin according to the supplier's instructions, and the beads were then washed with RNA-binding buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl). The coupling efficiency was 72%. The beads were equilibrated with washing buffer A [20 mM HEPES-KOH (pH 7.6), 40 mM KCl, 3 mM MgCl2, 2 mM dithiothreitol, 5% (vol/vol) glycerol] prior to use.

Affinity purification of C2 protein from NaCl extracts of MTs

The MT pellets were resuspended in the PIPES buffer containing 30% (vol/vol) glycerol and 0.5 M NaCl, and insoluble materials were removed by centrifugation at 10,000 g at 25°C for 5 min. The NaCl extracts were diluted about sevenfold with washing buffer A containing 100 U of RNasin/ml and incubated with the beads at 4°C for 90 min with gentle shaking. The beads were then collected using a magnet according to the supplier's instructions and washed eight times with washing buffer A containing 0.05% (wt/vol) Nonidet P-40 at 4°C for 5 min each with gentle shaking. After washing, the bound protein fractions were step-eluted with 200 μl of 0.1, 0.5, 1, and 2 M NaCl in washing buffer A containing 0.05% (wt/vol) Nonidet P-40. The eluted protein fractions were dialyzed against washing buffer A before being analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and EGMSA. The molecular mass standards used were as follows: bovine serum albumin (66 kDa), egg albumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.2 kDa).

Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

The MTs reassembled in the presence of GTP and taxol as described above were dissolved in the PIPES buffer containing 1 mM GTP, 20 μM Taxol, and 10% (vol/vol) glycerol, which were made to one-fourth of the original volume of the S200 fraction of brain cytoplasm. As the MT pellets were not easily solubilized under the conditions employed, insoluble aggregates were removed by centrifugation at 10,000 g for 5 min. Aliquots of the supernatant were incubated with 32P-BC1-1 RNA or 32P-D2 RNA in the PIPES buffer (100 μl) containing 1 mM GTP, 20 μM taxol, 100 U of RNasin/ml, and 10% (vol/vol) glycerol at 25°C for 20 min. The reaction mixtures were then overlaid on 20% (vol/vol) glycerol (800 μl) made with the PIPES buffer containing 1 mM GTP + 20 μM Taxol. After centrifugation at 200,000 g at 25°C for 30 min, the supernatant was removed carefully, and then the radioactivity in the pellet was determined by the Cerenkov method. The MT pellets were then suspended in the CaCl2 solution (100 μl), and aliquots of this suspension were electrophoresed under the conditions for EGMSA. The nucleotide sequence of D2 RNA is 5′-GGGGUUGGGGAUUUAGCUCAGGGGUUGGGCGCUU-3′. The underlined bases are different from those of BC1-1 RNA.

Estimation of the molecular mass of C2 protein

The 32P probe incubated with the required proteins was irradiated on ice for 5 min using a germicidal UV lamp (25 W) and separated electrophoretically under the gel shift assay conditions. Then, the gel was immersed in a buffer (375 mM Tris-HCl, pH 8.8, 0.1% SDS) for 15 min, and the UV-induced RNA-protein complexes were electroblotted onto Immobilon P membranes. The C1 and C2 complexes eluted from the membranes were digested with 1 mg RNase A/ml at 37°C for 15 min, followed by SDS-PAGE, and then detected by autoradiography.

Peptide sequence determination

Amino acid sequence analysis was performed according to the method of Aebersold et al. (1987). In brief, affinity-purified proteins were separated by SDS-PAGE and then blotted to ProBlott membranes, followed by Coomassie Brilliant Blue staining. The spot corresponding to the 42-kDa protein was excised and digested with 0.2 μg of lysylendopeptidase in 100 μl of 20 mM Tris-HCl (pH 8.5) and 8% (vol/vol) acetonitrile at 37°C overnight. The digested peptides were extracted with 8% (vol/vol) acetonitrile and purified by HPLC. The amino acid sequences of the recovered peptides were determined by a pulse liquid phase sequencer. Sequence homology was analyzed with the Genetyx computer program (Software Development Co., Tokyo, Japan).

Western blot analysis

Proteins separated by 10% SDS-PAGE were electroblotted to Immobilon P membranes. The blot was reacted with the required antibodies and detected using ProtBlot Western Blot AP Systems.

Immunoprecipitation analysis for protein—protein interaction

CaCl2 extract of MTs (100 μg) was mixed with 2.5 μg of pur α-specific antibody (rabbit anti-pur α 291-313 antibody) (Kelm et al., 1999) in a final volume of 250 μl and incubated for 60 min at room temperature. Goat anti-rabbit IgG (4.5 μg)-coupled magnetic Dynabeads were then added, and the mixtures were incubated for an additional 90 min. After incubation, the beads were captured with a magnet and washed five times (5 min each) with 25 mM HEPES (pH 7.5) and 150 mM NaCl. Bound protein was specifically eluted with a free multiple antigenic peptide (MAP-peptide) corresponding to amino acids 291-313 of pur α (20 μl at 85 μM) and then with 2% SDS, followed by western blot analysis using antibodies against pur proteins. Anti-pur α and anti-pur β were generous gifts from Dr. Robert J. Kelm. The MAP-peptide used was synthesized by the method of Tam (1988).

Purification of liver Translin

Purification was performed as described previously (Kobayashi et al., 1992). In brief, mouse liver S100 fraction was centrifuged at 2°C for 16 h, and the free RNP fraction obtained was dissolved in RNP buffer (50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, 5% glycerol, 0.25 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and then applied to a DE 52 column. After the column was washed with RNP buffer, proteins were eluted with a linear gradient of 0.05-0.5 M KCl in RNP buffer. Aliquots of eluted fractions were analyzed by EGMSA using 32P-labeled single-stranded BC1-CL1 DNA containing the binding sequences for Translin as a probe (Aoki et al., 1995). The active fractions (∼0.25 M KCl) were collected, dialyzed against 0.05 M sodium phosphate buffer (pH 7.5), and then applied to a hydroxylapatite column. Retained proteins were eluted with a linear gradient of 0.05-0.3 M sodium phosphate buffer (pH 7.5). The active fractions (∼0.09 M sodium phosphate) were pooled, dialyzed against RNP buffer, and then applied to a heparin-agarose column. After the column was washed with RNP buffer, proteins were eluted with a linear gradient of 0.05-0.5 M KCl in RNP buffer, and the active fractions were collected at ∼0.18 M KCl. These fractions were subjected to western blot analysis using anti-Translin antibody (Aoki et al., 1995).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

Figure 1a shows the location of two y-, h-elements and redundant copies of 14-nt G-, U-rich motifs (motifs I-III) in BC1 RNA (Kobayashi et al., 1998). Nucleotide sequences of motifs I-III and the putative dendrite-targeting motifs (Muslimov et al., 1997) are also shown in Fig. 1b. We first reinvestigated the trans-acting proteins that bind to these cis elements in an EGMSA using 32P-labeled subfragments of BC1 RNA (BC1 1-4; Fig. 1c) as probes. Consistent with our previous observations (Muramatsu et al., 1998), y-, h-element-containing probes (BC1-1 and BC1-3 RNAs) formed C1 complex, whereas 32P-BC1-1 and -2 RNAs additionally generated a distinct C2 complex (Fig. 2a). As the latter two RNA probes contain the putative dendrite-targeting motifs, which overlap G-, U-rich motifs I and II, the C2 protein may also be relevant to the dendritic translocation of BC1 RNA. The binding specificity of C2 protein to the 32P-BC1-1 was examined in a competition experiment (Fig. 2b). The unlabeled BC1-1 RNA reduced both the C1 and the C2 complexes, and the BC1-3 RNA, which contains the target site only for Translin protein (see Fig. 2a), reduced only the C1 complexes. In contrast, the amount of C2 complex was increased by the BC1-3 RNA (Fig. 2b). This increase appeared to result from competition of the BC1-3 RNA with the 32P-BC1-1 RNA for Translin and suggested that the binding site for Translin overlaps that for the C2 protein in BC1-1 RNA.

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Figure 2. Specific binding of C2 protein to the dendrite-targeting motifs. a: The binding site of C2 protein in BC1 RNA was analyzed in EGMSA. The 32P-RNA probes used were 32P-BC1-1 RNA (lane 1), 32P-BC1-2 RNA (lane 2), 32P-BC1-3 RNA (lane 3), and 32P-BC1-4 RNA (lane 4). b: The binding specificity of the C2 protein is shown. The 32P-BC1-1 RNA was used as the probe. Unlabeled competitor RNAs additionally included were as follows: no addition (lane 1), BC1-1 RNA (lanes 2 and 3), and BC1-3 RNA (lanes 4 and 5). Molar excesses of the unlabeled RNAs were 50 (lanes 2 and 4) or 200 (lanes 3 and 5). All lanes contained 4 μg of proteins from the glycerol gradient fractions containing BC1 RNP (tubes 15-17 in Fig. 3a).

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Next, we examined whether the C2 protein is contained in BC1 RNP by glycerol gradient centrifugation. The C2 protein again was detected with the 32P-BC1-1 RNA probe (Fig. 3a) but not with the 32P-BC1-3 RNA (Fig. 3b). Figure 3a also shows that this protein was heterogeneously distributed across the gradient. Note that the C2 protein was reduced in amount in fractions 15-17 in which the BC1 RNP containing Translin was distributed. This also appeared to result from an inhibitory effect of the Translin on the binding of C2 protein to the 32P-BC1-1 RNA in the EGMSA. These results suggested that unlike Translin the C2 protein is not a component of BC1 RNP but associates with some heterogeneous subcellular structure such as MTs in brain cells. Therefore, we examined whether the C2 protein can be precipitated with reassembled tubulin. The S200 fraction of brain cytoplasm was treated with GTP or GTP + Taxol, and the resulting MTs were sedimented by ultracentrifugation. The MT pellet obtained was dissolved in a CaCl2 solution and made up to the original volume of the S200 fraction; then, equivalent volumes of the CaCl2 extracts and the corresponding post-MT supernatant were analyzed using EGMSA to determine the amount of C2 protein. Figure 4a (top) shows that the C2 protein was enriched in the extracts of MTs formed with GTP (lane 2) as well as with GTP + Taxol (lane 3). This enrichment was abolished when an MT-disrupting reagent, colcemid, was included (lane 4), suggesting that the C2 protein associated with reassembled MTs. In support, the relative amounts of tubulin in the pellet determined by SDS-PAGE corresponded well to those of the C2 protein determined by EGMSA (Fig. 4a, middle). Next, we performed reassembly of MTs in the presence of increasing amounts of KCl to exclude the possibility that the association of C2 protein with MTs was due to physical entrapment, because dissociation from MTs at elevated ionic strength is a property shared by a variety of MT-associated proteins (Vallee and Collins, 1986; Elisha et al., 1995). Figure 4c (top) shows that the C2 protein in the MT pellet (lane 1) disappeared when KCl was added (lanes 2 and 3), but the formation of MTs was not affected (Fig. 4c, bottom). These results indicated that the association involved a salt-sensitive interaction and was not due to physical entrapment. Han et al. (1995a) reported that TB-RBP (Translin) can bind to MTs. However, in contrast to their observations, no C1 complexes containing Translin were detected in the CaCl2 extracts of the MT pellet (Fig. 4a, top). These results suggested that Translin was not able to bind to MTs. Consequently, high levels of C1 complexes remained in all of the supernatants (Fig. 4b, top). Western blot analysis using anti-Translin IgG also revealed that no Translin was co-precipitated with MTs (Fig. 4a, bottom), but similar amounts of Translin were detected in all of the post-MT supernatants (Fig. 4b, bottom). Consistent with the absence of Translin in the MT pellet, BC1 RNA was not detected either (data not shown). Thus, in contrast to the C2 protein, the association of BC1 RNP (BC1 RNA-Translin complex) with the MTs that were reassembled in vitro was not observed.

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Figure 3. Association of C2 protein with heterogeneously sized subcellular structures. An S100 fraction of brain cytoplasm was centrifuged on a glycerol gradient, and an aliquot of each alternate fraction was analyzed in EGMSA. The 32P-RNA probes used were 32P-BC1-1 RNA (a) and 32P-BC1-3 RNA (b).

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image

Figure 4. Specific association of C2 protein with MTs. Equivalent volumes of CaCl2 extracts of MT pellet (a) and post-MT supernatant (b) were analyzed by EGMSA (top), SDS-PAGE (middle), or western blotting using anti-Translin IgG (bottom). MTs were reassembled in the presence of 1 mM GTP (lane 2), 1 mM GTP + 20 μM Taxol (lane 3), or 1 mM GTP + 20 μM Taxol + 1 mM colcemid (lane 4). Lane 1, untreated control. KCl treatment (c) abolished the binding of C2 protein to MTs. Equivalent volumes of CaCl2 extracts of MTs assembled in the presence of 1 mM GTP + 20 μM Taxol (lane 1), 1 mM GTP + 20 μM Taxol + 300 mM KCl (lane 2), or 1 mM GTP + 20 μM Taxol + 500 mM KCl (lane 3) were analyzed by EGMSA (top) or SDS-PAGE (bottom). 32P-BC1-1 RNA was used as the probe in EGMSA. Only the relevant regions are shown.

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The binding specificity of the C2 protein precipitated with MTs to the 32P-BC1-1 RNA was examined (Fig. 5). The amount of C2 complex decreased as increasing amounts of unlabeled BC1-1 RNA were added (lanes 2 and 3), but no such effective competition was observed with BC1-3 RNA containing the target site for Translin only. Additionally, consecutive guanine residues present in the 5′ end region of BC1-1 RNA (nt 1-10; GGGGUUGGGG) appeared to constitute the core binding site, as poly(G) reduced the C2 complex (lane 8).

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Figure 5. The binding specificity of the C2 protein associated with the MT pellet. The 32P-BC1-1 RNA was incubated with CaCl2 extracts (0.1 μg) of MT pellet in the presence of unlabeled BC1-1 RNA (lanes 2 and 3), BC1-3 RNA (lanes 4 and 5), poly(A) (lane 6), poly(U) (lane 7), or poly(G) (lane 8). Molar excesses of the unlabeled subfragments of BC1 RNA were 20 (lanes 2 and 4) and 100 (lanes 3 and 5). The amount of homopolymer included was 10 ng.

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Although the ability of Translin (TB-RBP) to bind to MTs (Han et al., 1995a) was not reproduced under the conditions we employed (Fig. 4), our results suggested the specific interaction of the C2 protein with BC1 RNA and MTs and prompted us to investigate whether the C2 protein facilitates attachment of BC1-1 RNA to MTs. To address this issue, the binding experiments should be performed in the absence of Translin, as this protein exerted an inhibitory effect on the binding of C2 protein to the BC1-1 RNA (Figs. 2b and 3a). Therefore, for the analysis, we used the C2 protein-MT complexes that were reassembled from the S200 fraction of brain cytoplasm treated with GTP + Taxol, because these complexes contained virtually no Translin (Fig. 4a). We also used a 32P-labeled RNA (referred to as D2 RNA) as the negative control probe, which is identical to BC1-1 RNA except for three base changes in the G-, U-rich motif II (see Materials and Methods for the nucleotide sequences) and almost completely lacked the ability to bind to the C2 protein (Fig. 6c). Figure 6a shows that the amounts of the 32P-BC1-1 RNA that co-sedimented with the C2 protein—MT complexes upon centrifugation were about four times as much as those of the 32P-labeled D2 RNA that was precipitated under identical incubation and centrifugation conditions, suggesting that BC1-1 RNA was linked to the MTs through binding to the C2 protein. We also confirmed that the RNA that co-sedimented with the MTs was associated with the C2 protein. When the MT pellet that was dissolved in a CaCl2 solution was analyzed by PAGE, C2 complexes containing the 32P-BC1-1 RNA were detected (Fig. 6b). The 32P-BC1-1 RNA that was unable to penetrate into the gel might still be linked to the MTs (Fig. 6b, lane 1).

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Figure 6. Simultaneous binding of C2 protein to MTs and BC1-1 RNA. a: Preformed C2 protein-MT complexes were incubated with 32P-BC1-1 RNA (1) or 32P-D2 RNA (2) in the presence of 1 mM GTP + 20 μM Taxol. The reaction mixtures were then centrifuged, and the radioactivity in the pellet was determined. The radioactivity of 32P-D2 RNA (2) was 24% of control (CR) (1). b: The RNA-protein complexes precipitated with MTs [1 and 2 in (a)] were analyzed by PAGE. The RNA-protein complexes electrophoresed were as follows: lane 1, 32P-BC1-1 RNA-protein complex; lane 2, 32P-D2 RNA-protein complex. c:32P-D2 RNA generated no C2 complex with 5 μg of brain protein (lane 2). Lane 1 contained 32P-BC1-1 RNA and 5 μg of brain protein.

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Next, the molecular mass of C2 protein was estimated. Figure 7 shows that the C1 complex consisted of a 27-kDa protein (Translin protein), a 37-kDa protein, and a 43-kDa protein, whereas the C2 complex consisted only of a 43-kDa protein. Identical results were obtained by RNase T1 digestion (data not shown). We also confirmed that the C2 protein recovered from the MT pellet also generated the 43-kDa UV cross-linked adducts (lane 4). The small amount of 43-kDa protein found in the C1 complex (lane 2) may result from physical entrapment of the C2 complex by the C1 complex during electrophoresis. Additionally, the sequence specificity of the UV cross-linking was confirmed in a competition experiment (data not shown).

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Figure 7. Estimation of the molecular masses of the C2 proteins by UV cross-linking. C1 and C2 proteins cross-linked to 32P-BC1-1 RNA were separated by PAGE under the conditions for EGMSA and then transferred to Immobilon P membranes (lane 1). These complexes were analyzed by 10% SDS-PAGE after RNase A digestion. Molecular species consisted of C1 complex (lane 2), C2 complex (lane 3), or C2 complex obtained from the MT pellet (lane 4). Molecular masses of C1 proteins (43, 37, and 27 kDa) and C2 proteins (43 kDa) are indicated at the right.

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Next, we purified the C2 protein from the NaCl extracts of MTs using affinity beads bearing BC1-1 RNA. After extensive washing, the proteins that bound to the beads were step-eluted with NaCl (increasing concentrations), and EGMSA and UV cross-linking were performed to detect C2 protein. Consequently, the C2 complex and the 43-kDa UV cross-linked adducts co-eluted in the 0.5 M NaCl fraction (data not shown), suggesting that the C2 protein was purified by this RNA affinity chromatography. Therefore, in a separate experiment, we silver-stained aliquots of the purified protein fractions after SDS-PAGE. Proteins in the same fractions were also analyzed using EGMSA. Figure 8 shows that the majority of 32P-BC1-1 RNA-binding activity (C2 protein) was eluted from the beads in the 0.5 and 1 M NaCl fractions (top, lanes 4 and 5), and no activity was detected in the last wash fraction or the 0.1 or 2 M NaCl elution fractions (lanes 2, 3, and 6). In contrast to the heterogeneous population of proteins detected by SDS-PAGE in the unbound fraction or the 0.5 M NaCl elution fraction, a reduced number of proteins were detected in the 1 M NaCl elution fraction (Fig. 8, bottom, lane 5). Among them, a 42-kDa protein (arrow) closely matched the molecular mass of the C2 protein estimated by the UV cross-linking analysis (43 kDa). Therefore, this protein was subjected to microsequencing after lysylendopeptidase digestion. Consequently, amino acid sequences were obtained from two peptides, which contained 7 (RFYLDVK) and 10 (RFFFDVGSNK) amino acids. A computer search against the data base revealed that these two sequenced peptides were identical to amino acids 71-77 and 229-238 of pur α protein (Kelm et al., 1999). The pur α protein is a single-stranded DNA-and RNA-binding protein (Bergemann and Johnson, 1992; Herault et al., 1995) and has been implicated in control of DNA replication (Chang et al., 1996; Jurk et al., 1996) and transcription (Haas et al., 1995; Du et al., 1997; Kelm et al., 1997; Tretiakova et al., 1999a). Based on these observations, we next analyzed the C2 complex using anti-pur α-specific antibody (anti-α 291-313 antibody) (Kelm et al., 1999) in EGMSA. We also used pur β-specific antibody (anti-β 302-324 antibody) (Kelm et al., 1999) for confirmation, because the sequenced peptides were identical to amino acids 57-63 and 243-252 of pur β except for one amino acid change in the latter peptide (Ser236 of pur α is changed to Cys250 in pur β) (Kelm et al., 1999). Figure 9a shows that the C2 complexes were supershifted with both antibodies with similar efficiency (lanes 3 and 4), although anti-pur β also reduced the supershifted band more effectively than did anti-pur α (lanes 4 and 8). It was also shown that a 32P-end-labeled single-stranded cyclic AMP response element DNA probe (32P-ssCRE DNA) containing the target sequences for pur α (Kuo et al., 1999) formed similar complexes as well, which were supershifted with the antibodies (lanes 7 and 8). These results indicated that the C2 complex contains pur α and pur β proteins and suggested that these two pur isoforms form a heteromeric complex capable of binding to different forms of nucleic acid. Therefore, we next examined whether the pur α-specific antibody co-immunoprecipitates both pur α and pur β proteins. Consistent with the supershift experiment, Fig. 9b shows that the antibody was able to co-precipitate both proteins (lanes 4, 5, 9, and 10), providing direct confirmation of the presence of pur α/pur β heteromeric complexes in the C2 complex. These heteromeric structures seem prerequisite for nucleic acid binding of the pur proteins; however, the pur β may not be in direct contact with 32P-labeled BC1-1 RNA probe, as only the 43-kDa UV cross-linked adduct (pur α-RNA adduct) was detected in the C2 complex (Fig. 7).

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Figure 8. Affinity purification of C2 protein. NaCl extracts of the MT pellet were passed over affinity beads bearing BC1-1 RNA. Aliquots (1 μl) of dialyzed proteins at each purification step were analyzed in EGMSA using 32P-BC1-1 RNA as a probe (top), and aliquots (30 μl) of the same fractions were subjected to SDS-PAGE (bottom), followed by silver staining. Lane 1, flow-through fraction; lane 2, last wash fraction; lane 3, 0.1 M NaCl elution fraction; lane 4, 0.5 M NaCl elution fraction; lane 5, 1 M NaCl elution fraction; lane 6, 2 M NaCl elution fraction. Molecular mass standards are indicated at the right.

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image

Figure 9. Molecular characterization of the C2 protein present in the CaCl2 extract of MTs using anti-pur proteins. a: Supershift analysis of C2 complexes formed with 32P-BC1-1 RNA (lanes 1-4) or 32P-ssCRE DNA (lanes 5-8). Antibodies (0.5 μg) included no antibody (lanes 1 and 5), control rabbit IgG (lanes 2 and 6), anti-pur α antibody (lanes 3 and 7), and anti-pur β antibody (lanes 4 and 8). Arrows, supershifted bands. All lanes contained 0.1 μg of the CaCl2 extracts of MT pellet. The nucleotide sequence of ssCRE DNA probe used is 5′-CTGGGGGCGCCTCCTTGGCTGACGTCAGAGAGAGAG-3′ (Kuo et al., 1999). b: Analysis of molecular interactions between pur proteins by immunoprecipitation. Proteins bound to pur α-specific IgG (lanes 4, 5, 9, and 10) or control IgG (lanes 2, 3, 7, and 8) were eluted with a free MAP-peptide corresponding to pur α 291-313 (lanes 2, 4, 7, and 9) and then with SDS (lanes 3, 5, 8, and 10). Eluted proteins were separated by SDS-PAGE, followed by western blotting. IgGs used in the western blot analysis were anti-pur α 291-313 (lanes 1-5) and anti-pur β 302-324 (lanes 6-10). Protein extract from MTs with CaCl2 was similarly analyzed (lanes 1 and 6). The antibodies neutralized with 40 μg of MAP-peptides corresponding to pur α or pur β gave no specific bands (data not shown). The molecular masses of pur α (42 kDa) and pur β (39 kDa) are indicated, and only the relevant regions of the blot are shown.

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Next, the ability of these two pur proteins to bind to MTs was confirmed by western blot analysis (Fig. 10). Pur α and pur β co-precipitated with MTs from the S200 fraction of brain cytoplasm (lanes 3 and 5) were reduced in amount when colcemid was included in the reassembly reaction (lanes 4 and 6).

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Figure 10. Pur α and pur β proteins co-sedimented with reassembled MTs in vitro. Equivalent volumes of the CaCl2 extracts of MT pellet reassembled in the presence of 1 mM GTP (lanes 1, 3, and 5) or 1 mM GTP + 2 mM colcemid (lanes 2, 4, and 6) were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue staining (lanes 1 and 2) or western blotting using anti-pur α antibody (lanes 3 and 4) or anti-pur β antibody (lanes 5 and 6). The molecular masses of pur α (42 kDa) and pur β (39 kDa) are indicated, and only the relevant regions of the blot are shown.

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Based mainly on several recent findings (Han et al., 1995a; Wu et al., 1997; Kobayashi et al., 1998; Muramatsu et al., 1998), we have suggested that BC1 RNA is translocated to the neuronal dendrites along MTs in the complex with Translin. Nevertheless, it does not seem possible to ascribe a role to Translin in linking BC1 RNA to MTs, as, in contrast to Han et al. (1995a), no association of Translin with MTs was observed (Fig. 4a and b). The reason for this discrepancy is unclear, but it appears to be relevant to the conclusion made by Wu et al. (1997) that TB-RBP is the mouse homologue of Translin. They claimed that TB-RBP protein showed testis- and brain-specific expression (Han et al., 1995b), but we have also detected similar levels of 32P-BC1-1 RNA-binding activity (C1 complex) in the S100 fraction of liver and kidney cytoplasm as well as brain cytoplasm using EGMSA (our unpublished observations). Although the presence of Translin in these C1 complexes was also demonstrated with anti-Translin IgG (data not shown), we purified the protein (liver Translin) from the S100 fraction of liver cytoplasm by a combination of column chromatography, including DE 52, hydroxylapatite, and heparin—agarose (Kobayashi et al., 1992). Brain Translin protein was also purified in parallel from comparable amounts of the S100 fraction of brain cytoplasm to compare the relative amounts of Translin proteins in the two tissues. Figure 11 shows that both the brain and liver Translin proteins were eluted from the heparin column at similar KCl concentrations (∼ 180 mM KCl) and detected by western blot analysis using anti-Translin IgG. These results together indicated that Translin protein is not specific to brain and testis and in turn suggested that Translin protein is widely expressed in many tissues. It should be noted that Translin mRNA has been detected in many tissues (Aoki et al., 1995; Gu et al., 1998). Considering all results, the conclusion made by Wu et al. (1997) seems inadequate in that the tissue distribution of the TB-RBP and the Translin was not the same.

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Figure 11. Purification of liver Translin by DE 52, hydroxylapatite, and heparin-agarose column chromatography. Only the western blots of the Translin protein purified on a heparin-agarose column are shown. Both the brain Translin (top) and the liver Translin (bottom) were eluted at ∼180 mM KCl.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

Pur α binds to the DNA and RNA sequences containing repeats of GGN (Bergemann and Johnson, 1992; Gallia et al., 1999) and has been known as the nuclear factor that plays regulatory roles in both transcription and replication (Haas et al., 1995; Chang et al., 1996; Jurk et al., 1996; Du et al., 1997; Kelm et al., 1997; Tretiakova et al., 1999a). However, no cytoplasmic functions have so far been reported, despite its high level of expression in the cytoplasm of neuronal cells (Osugi et al., 1996; unpublished observations, 1999). In this study, we suggested that pur α and its isoform, pur β, may mediate the dendritic translocation of BC1 RNA. This novel cytoplasmic role of the pur proteins was suggested by the observations that they had dual ability to bind to both RNA and MTs and attached naked BC1 RNA to MTs through binding to the dendrite-targeting RNA motifs (Figs. 4, 6, 9, and 10). However, we have also made several unsuccessful attempts to link the BC1 RNP (BC1 RNA—Translin complex) to preformed pur protein—MT complexes (data not shown). It seems likely the binding of pur proteins to the BC1 RNP was inhibited by the Translin present within the RNP, due to the fact that the mode of binding of these two classes of proteins to BC1-1 RNA was mutually exclusive (Figs. 2 and 3). Therefore, at the moment, we do not have a good explanation of how the pur proteins interact with the target BC1 RNA sequences (dendrite-targeting motifs) to link the BC1 RNP itself to MTs. However, the activities and specificity of these RNA-binding proteins may be changed by posttranslational modifications such as phosphorylation and/or dephosphorylation. In this respect, the presence of multiple amino acid residues in Translin was demonstrated, each of which could be phosphorylated (Aoki et al., 1995). The binding of Translin to a single-stranded DNA probe was suppressed by treatment with protein phosphatase (Taira and Baraban, 1997). Acid phosphatase treatment of TB-RBP also abolished the RNA-binding activity (Kwon and Hecht, 1993), although the relationship between TB-RBP and Translin is obscure. Furthermore, we recently observed that the RNA-binding activities of pur proteins and Translin were significantly reduced by alkaline phosphatase treatment (data not shown). Therefore, phosphorylation and/or dephosphorylation of these two classes of proteins resulting in the modulation of their RNA-binding activities or specificity could enable the pur proteins to bind to the BC1 RNA in place of the Translin within the RNP. As pur proteins did not appear to be components of BC1 RNP but associated with MTs (Figs. 3, 4, and 10), they may transiently interact with the RNP during transport. In this context, it would be interesting to speculate that such transient binding of pur proteins to the BC1 RNA may be regulated by changes in the phosphorylation state of pur proteins as well as Translin in response to synaptic activity, because the BC1 RNA transport to neuronal dendrites was reportedly activity dependent (Muslimov et al., 1998).

It is also possible that the pur proteins may associate with the BC1 RNP via protein—protein interactions with the Translin, as pur α has been reported to interact with several cellular and viral proteins to perform diverse functions (Johnson et al., 1995; Gallia et al., 1999; Tretiakova et al., 1999a,b). Moreover, it was also reported that combinatorial interactions between pur α, pur β, and a Y-box protein, MSY1, may have functional significance in regulating transcriptional activity of the smooth muscle α-actin gene (Kelm et al., 1999). Such homo- and heteromeric interactions may also be facilitated upon phosphorylation of the proteins involved, as described by Tretiakova et al., (1999a).

In the present study, we also demonstrated that the dendrite-targeting motifs in BC1 RNA are not the binding site for one protein only but were bound by two different classes of proteins (pur proteins and Translin) differing in RNA-binding specificity (Figs. 2 and 5) and that they associated with different subcellular structures (MTs or BC1 RNP). These observations suggest that these proteins co-operate in the transport of BC1 RNA along dendritic MTs. However, it seems unlikely the motifs are involved solely in the translocation of BC1 RNA along MTs. Rather, it is conceivable that these motifs could also mediate other multiple steps in the process of BC1 RNA transport by interacting with distinct RNA-binding proteins. Although the goal of BC1 RNP in dendrites is not known, several investigators have reported that BC1 RNA is particularly concentrated in synaptodendrosomes (Chicurel et al., 1993; Rao and Steward, 1993). Furthermore, emerging evidence suggests that proteins are synthesized at synapses (Kang and Schuman, 1996; Weiler et al., 1997; Steward et al., 1998; Wu et al., 1998). Together, the BC1 RNP carrying several translational regulators (Kremerskothen et al., 1998; Muramatsu et al., 1998) may ultimately be anchored near synapses within the dendritic spines. Thus, it seems possible that the pur proteins may mediate the translocation and anchoring of BC1 RNA within these postsynaptic dendritic compartments. However, as described above, it is also possible that other dendrite-targeting motif-binding proteins may be present in association with components of the cytoskeleton abundant in dendritic spines, such as actin (Fifkova and Morales, 1992) or α-actinin-2 (Wyszynski et al., 1997). Actin filaments are reportedly important for anchoring of the message at the cortex of Xenopus oocytes (Yisraeli et al., 1990). It would be interesting to speculate that such a putative actin-binding protein may also interact with pur α, pur β, or Translin in the transport and anchoring processes.

Finally, as the dendrite-targeting motifs are well conserved in MAP2a/b mRNA (Muslimov et al., 1997; see also Fig. 1), the pur proteins may also play a role in the dendritic transport of a subset of mRNAs. In this respect, it would be intriguing to consider whether the pur proteins bind to other known dendritic mRNAs (Steward et al., 1995).

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Preparation of RNA probes
  5. Electrophoretic gel mobility shift assays (EGMSAs)
  6. Preparation of RNA affinity beads
  7. Binding of 32P-BC1-1 RNA probes to preformed C2 protein-MT complexes
  8. RESULTS
  9. DISCUSSION
  10. Acknowledgements

We thank Dr. Robert J. Kelm, Jr., of the Mayo Clinic/Foundation for the generous gifts of anti-pur α and anti-pur β antibodies. We also thank Dr. M. Kasai of the National Institute of Infectious Diseases for the anti-Translin IgG.

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