Post-transcriptional regulation of the GAP-43 gene by specific sequences in the 3' untranslated region of the mRNA - PubMed (original) (raw)

Post-transcriptional regulation of the GAP-43 gene by specific sequences in the 3' untranslated region of the mRNA

K C Tsai et al. J Neurosci. 1997.

Abstract

We have shown previously that GAP-43 gene expression during neuronal differentiation is controlled by selective changes in mRNA stability. This process was found to depend on highly conserved sequences in the 3' untranslated region (3' UTR) of the mRNA. To map the sequences in the GAP-43 3' UTR that mediate this post-transcriptional event, we generated specific 3' UTR deletion mutants and chimeras with the beta-globin gene and measured their half-lives in transfected PC12 cells. Our results indicate that there are two distinct instability-conferring elements localized at the 5' and 3' ends of the GAP-43 3' UTR. Of these destabilizing elements, only the one at the 3' end is required for the stabilization of the mRNA in response to treatment with the phorbol ester TPA. This 3' UTR element consists of highly conserved uridine-rich sequences and contains specific recognition sites for two neural-specific GAP-43 mRNA-binding proteins. Analysis of the levels of mRNA and protein derived from various 3' UTR deletion mutants indicated that all mutants were translated effectively and that differences in gene expression in response to TPA were attributable to changes in GAP-43 mRNA stability. In addition, the phorbol ester was found to affect the binding of specific RNA-binding proteins to the 3' UTR of the GAP-43 mRNA. Given that, like the GAP-43 mRNA, its degradation machinery and the GAP-43 mRNA-binding proteins are expressed primarily in neural cells, we propose that these factors may be involved in the post-transcriptional regulation of GAP-43 gene expression during neuronal differentiation.

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Figures

Fig. 1.

Fig. 1.

Schematic representation of the wild-type GAP-43 cDNA and of several 3′ UTR deletion mutants and chimeras.A, The restriction map shown in the wild-type construct (WT) corresponds to the full-length rat GAP-43 cDNA (GA11B; Neve et al., 1987) that was used to generate the various constructs. Segments represent the different fragments created by restriction enzyme digestion. Chimeras were generated by a combination of these fragments with the β-globin coding region. B, Sequence of the GAP-43 3′ untranslated region (3′ UTR) of the GAP-43 cDNA. The sequences in the B region are indicated in_italics_, and the three regions in U3, including five point mutations, are underlined.

Fig. 2.

Fig. 2.

Role of the GAP-43 3′ UTR on mRNA stability. PC12–N36 cells were transfected with a rat full-length GAP-43 cDNA construct (WT), a deletion mutant without the 3′ UTR (Δ3′ UTR), or chimeras with the β-globin cDNA (C1 and C2; see Fig. 1). Cells were incubated with cadmium to induce high levels of transfected mRNAs. For mRNA decay studies, RNAs were isolated from cells harvested at the time points indicated after removal of cadmium. Control lanes (Co) show the basal levels of the transfected mRNA in the absence of the metal. A, Northern blots from representative mRNA decay experiments: analysis was performed with 15 μg of cytosolic RNA. The same membrane was probed for GAP-43 first and then reprobed for G3PD. B, mRNA decay curves. GAP-43 mRNA levels were determined by densitometry, corrected by those of G3PD, and expressed relative to those before the decay phase (time 0).

Fig. 3.

Fig. 3.

Analysis of the stability of the different GAP-43 3′ UTR deletion mutants and chimeras with the β-globin cDNA in transfected cells. PC12–N36 cells were transfected with various constructs in the expression vector pMEP4, and mRNA decay studies were performed as indicated in Figure 2. Northern blots show the rate of decay of different GAP-43 3′ UTR deletion mutants and chimeras. Blots were probed with either GAP-43 (A) or β-globin (B) cDNAs and reprobed with G3PD to control for RNA loading. The structure of each construct is shown in Figure 1.

Fig. 4.

Fig. 4.

Effect of three U-rich regions in GAP-43 3′ UTR and the region surrounding the stop codon on GAP-43 mRNA stability.A, Northern blots show the decays of the wild-type and mutant U3 and D40 constructs.B, mRNA decay curves indicate that neither the five U by A replacements in U3 nor the deletion of 40 nt in _D40_has any effect in the rate of decay of the mRNA.

Fig. 5.

Fig. 5.

Analysis of the stability of the wild-type GAP-43 mRNA and several 3′ UTR mutants in PC12–N36 cells. mRNA decay assays were performed in stably transfected PC12–N36 cells, as described in Figure 2. The relative levels of the GAP-43 mRNA at different decay times were calculated by densitometric analysis of Northern blots and were corrected by the levels of G3PD in the same sample. The plot shows representative decay curves for the wild-type GAP-43 mRNA (WT), the AC, AB, and A mutants, and the_Globin_-B and_Globin_-C chimeras.

Fig. 6.

Fig. 6.

TPA causes the induction and stability of the GAP-43 mRNA in transfected PC12–N36 cells. A, Northern blots demonstrate the levels of induction and stability of the transfected wild-type GAP-43 mRNA in PC12–N36 cells. For the induction phase, cells were treated for 16 hr in the presence of NGF (NGF; 100 ng/ml), TPA (TPA; 160 n

m

), or CdCl2 (Cd; 5 μ

m

). For the decay phase, after cadmium induction, cells were washed out of the metal ion and incubated for 6 hr in the presence (+DRB) or absence of DRB (60 μ

m

) and_TPA_ (160 n

m

) or polymyxin B (PB; 2000 U/ml). B, Cells were induced for 16 hr with either Cd2+ (filled squares) or TPA and Cd2+ (open squares), and mRNA decay assays were performed in the presence or absence of 160 n

m

TPA. The relative half-lives of the mRNA were calculated as described in Materials and Methods.

Fig. 7.

Fig. 7.

Effect of TPA on GAP-43 mRNA and protein levels in PC12–N36 cells transfected with different 3′ UTR mutants.A, B, Northern (A) and Western (B) blots show GAP-43 mRNA and protein levels in permanently transfected control cells (Co) or in cells induced for 16 hr with cadmium (Cd) or phorbol ester (TPA), as described in Figure 6. The levels of G3PD in Northern blots and tubulin in Western blots are presented as controls for gel loading. C, Relative induction of the GAP-43 mRNA and protein in TPA-treated PC12–N36 cells. The levels of induction of each construct represent the mean of at least three independent experiments.

Fig. 8.

Fig. 8.

Comparative analysis of the half-life of the GAP-43 mRNA in COS-7 cells. Northern blots show the decay of the transfected wild-type GAP-43 mRNA in PC12–N36 cells and non-neural COS-7 cells. The half-life of the mRNA was found to be ∼2.5-fold shorter in the neural lines than in COS-7 cells.

Fig. 9.

Fig. 9.

Detection of GAP-43 mRNA-binding proteins by UV cross-linking experiments. A, 32P-labeled RNAs containing the entire GAP-43 3′ UTR (GAP/3′) or the B region (GAP/B) were synthesized in vitro, as described by Kohn et al. (1996a). RNAs (0.5 ng, 5 × 104 cpm) were incubated with 50 μg of brain S100 protein for 10 min at 4°C. To test for binding specificity, we incubated reactions in the presence or absence of a 100-fold excess of the corresponding cold competitor RNA (GAP/3′ or_GAP/B_). After UV irradiation, RNA protein complexes were analyzed in 10% polyacrylamide gels. B, RNA-binding reactions were performed with 32P-labeled_GAP/3′_ RNA and cytosolic extracts derived from control PC12 cells (Co) or cells induced for 16 hr with 160 n

m

TPA (+TPA). Gels were exposed to film for 3–7 d at −80°C.

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