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Table 1. Biochemical purification and peptide sequence analyses reveal that VP-RBP is the rat PABP

Protein purification

Peptide sequence

Percent identity to mouse PABP1

Heparin column: 0.2 M NaCl

Affinity purification: 0.1% SDS

P-I: EFSPFGTITSAK

100%, amino acid, 313–324

Heparin column: 0.5 M NaCl

Affinity purification: 1 M NaCl

P-II: GYGFVHFETQEAAER

100%, amino acid, 139–153

Heparin column: 0.5 M NaCl

Affinity purification: 0.1% SDS

P-III: NFGEDMDDERL

100%, amino acid, 197–207

(S-90) was carefully removed. Solid ammonium sulfate was added to a final concentration of 45% saturation at 0°C. If necessary, the pH was adjusted to 7.8 with 1 M Tris-base. Precipitated proteins were sedimented for 30 min at 3,500×g. The supernatant fractions were discarded. Proteins were dissolved in 18 ml of 10 mM Hepes, pH 7.8/10 mM NaCl/2 mM DTT/1 mM EDTA/2% glycerol (vol/vol)/0.5 mM PMSF and desalted by using the same buffer and a 5-ml HiTrap desalting column (Amersham Pharmacia Biotech), according to the manufacturer’s instructions. Typically, the desalted eluent had a concentration of ˜7 mg/ml of protein, as determined with the Protein Assay Reagent (Bio-Rad) and BSA as a standard. Twenty-five milligrams of protein was subjected to heparin column chromatography (5 ml of HiTrap heparin column, Amersham Pharmacia Biotech). Proteins were eluted with 10.5 ml each (7 fractions, 1.5 ml each) of 10 mM Hepes, pH 7.8/2 mM DTT/1 mM EDTA/2% (vol/vol) glycerol/0.5 mM PMSF containing 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl, respectively. Protein fractions were snap-frozen in liquid nitrogen and stored at –80°C. Each fraction (1.5 µl) was tested for VP mRNA-binding activity by performing U V-crosslinking assays, as described (18). Fractions containing binding activity were further purified by affinity chromatography.

Affinity Chromatography. Preparation of biotinylated VP mRNA. Full-size VP mRNA was prepared by using 5 µg of linearized template DNA and the RiboMAX T7-system (Promega) in a 100-µl assay. Biotin-16-UTP (Boehringer Mannheim) was included at a final concentration of 0.3 mM. In vitro transcripts were purified by using the RNeasy midi kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.

Coupling of biotinylated RNA to streptavidin-coated paramagnetic particles. Streptavidin-coated paramagnetic particles [0.6 ml (1 mg/ml); Promega] was washed three times with 1 ml each of PBS (10 mM Na-phosphate, pH 7.4/150 mM NaCl). Three hundred picomol of biotinylated VP RNA in 1 ml of PBS was incubated with the particles for 30 min at room temperature on a rotating wheel. A coupling efficiency of 20–30% was routinely achieved. After removal of the RNA solution, the beads were washed twice with 1 ml of PBS and twice with 1 ml of binding buffer [10 mM Tris-HCl, pH 7.8/2 mM DTT/1.5 mM EDTA/10 mM KCl/4% (vol/vol) glycerol/6.7 µg/µl yeast tRNA] and Complete protease inhibitor.

Affinity chromatography. Fractions obtained during heparin column chromatography containing binding activity were desalted as described by using binding buffer. For analytical affinity purification, 1.5–2.0 ml of desalted protein fractions (˜1 mg of protein) was incubated with biotinylated VP RNA coupled to 0.6 ml of streptavidin-coated paramagnetic particles for 20 min at room temperature after addition of heparin (final concentration 2.5 mg/ml) on a rotating wheel. Protein solution (unbound proteins) was removed and saved for later analysis. The particles were washed (2 min each) once with 1 ml of binding buffer containing heparin (2.5 mg/ml), twice with 1 ml each of binding buffer, and once with 200 µl of binding buffer. Bound protein was eluted with 100 µl of 0.1% SDS for 10 min at room temperature and stored at—20°C. In some cases, proteins were eluted first with the same volume of 1 M NaCl/10 mM Tris-HCl, pH 7.8/1.5 mM EDTA/2 mM DTT and Complete protease inhibitor for 30 min at room temperature, concentrated 10-fold by using Vivaspin columns (Sartorius), snap-frozen in liquid nitrogen, and stored at –80°C. For preparative purposes, affinity purification assays were scaled up 10-fold.

Peptide Sequencing. Proteins purified by affinity chromatography were separated by SDS/PAGE and stained for several hours with colloidal Coomassie blue (Roti-Blue, Roth, Karlsruhe, Germany) according to the protocol recommended by the manufacturer. The protein bands were cut out, washed in water (3×2 h), and treated with acetonitrile for 30 min. The shrunken gel pieces were rehydrated by addition of 1 µg of endoproteinase LysC (Roche Molecular Biochemicals) in 100 µl of digestion buffer (50 mM Tris-HCl, pH 8.5/1 mM EDTA) and incubated overnight at 37°C. The reaction was stopped by adding 1 µl of trifluoroacetic acid (TFA), and the supernatant fraction was collected. The gel pieces were sequentially incubated for 1 h with 100 µl each of reaction buffer, TFA/acetonitrile (50:50, vol/vol), and acetonitrile. All solutions were combined, and the proteolytic fragments were separated by narrowbore HPLC (130A, Applied Biosystems) on a C4 reverse-phase column (Vydac C4, 300 A pore size, 5 mm particle size, 2.1×250 mm). Peptides were eluted with a linear gradient (0–100% B in 50 min; solvent A: water/0.1% TFA, solvent B:70% acetonitrile/0.09% TFA) at a flow rate of 200 ml/min. Peptide-containing fractions detected at 210 nm were collected into siliconized tubes and frozen immediately.

Peptide sequences (Table 1) were determined by standard Edman degradation on an automatic sequencer (476A, Applied Biosystems).

Cloning of Rat Poly(A)-Binding Protein (PABP). A fragment of the rat PABP cDNA was amplified by the PCR after reverse transcription of rat brain RNA. Two fully degenerate primers were designed: forward primer, 5'-TT[TC]GT[GATC]CA[TC]TT-[TC]GA[GA]AC[GATC]CA[GA]GA[GA]GC-3', deduced from the amino acid sequence FVHFETQEA of peptide P-II, and reverse primer, 5'-[GAT]AT[GATC]GT[GATC]-CC[GA]AA[GATC]GG[GATC]GA[GA]AA[TC]TC-3', deduced from the amino acid sequence EFSPFGTI of peptide P-I.

Table 2. Comparison of axonal and dendritic VP mRNAs

Axonal VP transcripts

have shorter poly(A) tails than transcripts located in the perikarya

are transported to axons after translation

are located in varicosities devoid of peptide hormones

are not associated with ribosomes and are therefore not translated.

Dendritic VP transcripts

are identical in size to transcripts located in the perikarya

are transported to dendrites before translation

are located in parts of dendrites that contain ribosomes and small cisterns of rough endoplasmic reticulum

are most likely translated on-site.



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