following preparations were processed for protein identification: (i) Proteins eluted with 0.2 M NaCl from a heparin column, affinity purified and eluted with 0.1% SDS; (ii) proteins eluted with 0.5 M NaCl from a heparin column, affinity purified and eluted with 1 M NaCl; and (iii) proteins eluted with 0.5 M NaCl from a heparin column, affinity purified and eluted with 0.1% SDS. Proteins were digested separately with endoproteinase Lys-C. The resulting peptides were resolved by HPLC. The elution profiles were very similar, indicating that the proteins are either identical or at least highly related (data not shown). One peptide from each chromatographic separation (but exhibiting different retention times) was subjected to sequence analysis, revealing that the 78-kDa protein is the rat PABP. All peptides are 100% identical to parts of mouse PABP1 (Table 1). Cloning and cDNA sequencing showed that the rat PABP consists of 636-aa residues with a high degree of identity when compared with mouse (99.7%; ref. 19) and human (99.5%; ref. 30) PABP. Hence, VP-RBP will be referred to as PABP.
We do not know why PABP elutes from the heparin column with 0.2 and 0.5 M NaCl, but several explanations are possible. First, covalent modifications, for example phosphorylated forms, of PABP with reduced affinity for the negatively charged ligand could exist. Second, PABP may be part of larger complexes displaying distinct binding characteristics to heparin. Currently, we cannot discriminate between these possibilities. However, UV-crosslinking competition analyses performed with VP mRNA indeed reveal different binding behaviors of PABP in cytosolic extracts and in partially purified protein pools (Fig. 7). PABP present in rat brain cytosolic extracts exhibits the highest degree of binding specificity. Complex formation is inhibited by a molar excess of unlabeled VP mRNA but not by a-tubulin transcripts and segments spanning various parts of the SSTR3 mRNA. With ribohomopolymer competitors, poly(A) competed as would be expected, whereas poly(U), poly(G), and poly(C) were inefficient (Fig. 7 A). When the same series of experiments was done with proteins eluted with 0.2 M NaCl from a heparin column, additional, albeit minor, competition was observed with one of the SSTR3 RNA segments (SSTR-b probe) and with poly(G) (Fig. 7 B). The lowest degree of specificity with respect to interaction with VP mRNA is observed for PABP present in the protein pool eluted with 0.5 M NaCl from the column (Fig. 1 C). These data clearly demonstrate that binding specificity of PABP to VP mRNA is determined by additional parameters, for instance a covalent modification or by other proteins that could alter PABP’s sequence selection by protein/protein interactions. Apparently, this “specificity factor,” whatever its nature, is brain specific. Evidence stems from UV-crosslinking analyses shown in Fig. 3 B. Even though PABP is known to be extremely abundant (31), most peripheral tissues and non-neuronal cell lines harbor much lower amounts of the protein in a form that is able to interact with VP mRNA compared with brain tissue.
Possible Role of PABP in VP mRNA Metabolism. PABP harbors four highly conserved RNA recognition motifs (RRM; 80–100 aa in length) at the N-terminal part of the protein and a more divergent C-terminal auxiliary domain (32). It binds with high affinity to the poly (A) tail of mRNAs, thereby enhancing translation via interaction with initiation factors bound at the 5' end (33). Furthermore, it stabilizes mRNAs in a translation-dependent manner (34). Binding studies with individual RRMs or combinations thereof revealed several interesting features: single RRMs are unable to bind to poly (A). RRMs 1 and 2 have a high affinity for poly(A) identical to that of the full-size protein, whereas RRMs 3 and 4 have a much lower affinity for poly(A). In fact, binding of RRMs 3 and 4 to poly(U)- and poly(G)-sequences is much better than to poly(A) (35, 36). In yeast, PABP is essential for cell viability. Yet, whereas deletion of RRMs 1 and 2 alone still supported growth, removal of RRM 4 joined with C-terminal amino acid residues did not, suggesting critical functions of this sequence (35). Taken together, RRMs 1–4 are functionally diverse, and features other than high-affinity binding to poly(A) sequences are essential for cell viability. PABP is an extremely abundant protein. HeLa cells, for instance, have a roughly 3-fold excess of protein over binding sites on poly(A) mRNAs (31). Because PABP interacts with sequences other than poly(A) in vitro (31, 36), it probably has additional functions in mRNA metabolism. For instance, PABP is able to control translation of its own mRNA, and it does so by specific association with sequences of the 5'-UTR (37, 38).
Given the heterogeneous roles of PABP, it is conceivable that it may be involved in regulating the translational state of the VP (and possibly of other) mRNA. Accumulating evidence suggests that dendritically localized mRNAs are not translated until external stimuli trigger the activation of protein synthesis (5, 39). Translational silencing by PABP could, for instance, be accomplished by its binding to the DLS of the VP mRNA. A direct or indirect interaction of this (or these) molecule(s) with those that are bound to the poly(A) tract could inhibit translational stimulation, because it interferes with the interaction of poly(A) tail-bound PABP with translational initiation factors at the 5'-end of the mRNA. A similar model involving PABP as part of a larger and preexisting protein complex has been proposed as a mechanism that regulates translation-dependent turnover of the c-fos mRNA (40).
Several questions to be addressed in future experiments remain open, (i) Which RRMs of PABP participate in its interaction with DLS of the VP mRNA? (ii) What type of molecule (or modification) determines its binding specificity to the DLS? (iii) Does PABP also play a role in the metabolism of other dendritically localized mRNAs, and what exactly is that role?
We thank Susanne Franke for expert technical assistance. This work is supported by the Deutsche Forschungsgemeinschaft and the Volkswagenstiftung (to D.R. and E.M.). Part of this work forms the Ph.D. thesis of Carola Fuhrmann.
1. Steward, O. (1997) Neuron 18, 9–12.
2. Kuhl, D. & Skehel, P. (1998) Curr. Opin. Neurobiol. 8, 600–606.
3. Mohr, E. (1999) Prog. Neurobiol. 57, 507–525.
4. Tiedge, H., Bloom, F.E. & Richter, D. (1999) Science 283, 186–187.
5. Schuman, E.M. (1999) Neuron 23, 645–648.
6. Alvarez, J., Giuditta, A. & Koenig, E. (2000) Prog. Neurobiol. 62, 1–62.
7. Bashirullah, A., Cooperstock, R.L. & Lipshitz H.D. (1998) Annu. Rev. Biochem. 67, 335–394.
8. Barbarese, E., Brumwell, C., Kwon, S., Cui, H. & Carson, J.H. (1999) J. Neurocytol. 28, 263–270.
9. Gonzalez, I., Buonomo, S.B.C., Nasmyth, K. & von Ahsen, U. (1999) Curr. Biol. 9, 337–340.
10. King, M.L., Zhou, Y. & Bubunenko, M. (1999) BioEssays 21, 546–557.
11. Lipshitz, H.D. & Smibert, C.A. (2000) Curr. Opin. Genet. Dev. 10, 476–488.
12. Blichenberg, A., Schwanke, B., Rehbein, M., Garner, C.C., Richter, D. & Kindler, S. (1999) J. Neurosci. 19, 8818–8829.
13. Prakash, N., Fehr, S., Mohr, E. & Richter, D. (1997) Eur. J. Neurosci. 9, 523–532.
14. Mori, Y., Imaizumi, K., Katayama, T., Yoneda, T. & Tohyama, M. (2000) Nat. Neurosci. 3, 1079–1084.
15. Muslimov, I.A., Santi, E., Hamel, P., Perini, S., Higgins, D. & Tiedge, H. (1997) J. Neurosci. 17, 4722–4733.
16. Rehbein, M., Kindler, S., Horke, S. & Richter, D. (2000) Mol. Brain Res. 79, 192–201.
17. Monshausen, M., Putz, U., Rehbein, M., Schweizer, M., DesGroseillers, L., Kuhl, D., Richter, D. & Kindler, S. (2001) J. Neurochem. 76, 155–165.
18. Mohr, E., Fuhrmann, C. & Richter, D. (2001) Eur. J. Neurosci., 13, 1107–1112.
19. Wang, M., Cutler, M., Karimpour, I. & Kleene, K.C. (1992) Nucleic Acids Res. 20, 3519.