ElrA and AUF1 differentially bind cyclin B2 mRNA - PubMed (original) (raw)

ElrA and AUF1 differentially bind cyclin B2 mRNA

Xun Guo et al. Biochem Biophys Res Commun. 2008.

Abstract

In Xenopus embryos, maternal cyclins drive the first 12 cell divisions after which several cyclins are terminally degraded, including cyclin B2. Cyclin B2 disappearance is due to transcription-mediated mRNA deadenylation at the midblastula transition, when transcription initiates and the cell cycle lengthens. To further define the mechanism, we characterized proteins capable of binding cyclin B2 3'UTR. We show that ElrA and AUF1 compete for binding to regions containing cytoplasmic polyadenylation elements (CPEs), with AUF1 binding increasing at the midblastula transition. Deletion of both CPEs abrogates polyadenylation but has no effect on deadenylation or binding of ElrA or AUF1. Overexpression of ElrA or AUF1 does not alter cyclin B2 mRNA stability. These results show that ElrA and AUF1 bind to cyclin B2 mRNA independent of CPEs and function by binding other elements.

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Figures

Figure 1

Figure 1. Endogenous ElrA and AUF1 bind cyclin B2 3′UTR

A, UV-crosslink analysis of 4 and 8h embryos with radiolabeled cyclin B2 3′UTR in the presence of 0, 1, 5, or 10 molar excess unlabeled cyclin B2 3′UTR or cyclin A1ΔARE. Arrow, 37-kDa protein competed by B2 3′UTR. Arrowhead, proteins not competed. Double arrowheads, proteins competed by both. B2, cyclin B2 3′UTR; B2R, cyclin B2 3′UTR+RNase. B, Immunoblot of unfertilized eggs (U) through 10 hpf embryos sequentially probed with ElrA and AUF1 antibodies. Top numbers, hours postfertilization. C, UV-crosslink of 4 and 8h embryos with radiolabeled cyclin B2 3′UTR or A1ΔARE (A1) followed by immunoprecipitation with IgG, αElrA or αAUF1. Input, UV-crosslink before immunoprecipitation. Arrowheads, immunoprecipitated protein. D, RNA-pulldown assay of 4 and 8h embryos with biotinylated cyclin B2 3′UTR; followed by immunoblotting for AUF1, ElrA, and CPEB (Pulldown). Input, immunoblot of embryos used for the pulldown. 8+a, embryos injected with α-amanitin. E, RNA-pulldown of 4 and 8h embryos with biotinylated cyclin B2 3′UTR or A1ΔARE; followed by immunoblotting for AUF1, ElrA, and actin. Markers are in kDa for A-C; n=3 for A, C-E; n=5 for B.

Figure 2

Figure 2. ElrA and AUF1 bind cyclin B2 3′UTR in vitro

A, UV-crosslink analysis of GST-ElrA and GST-AUF1 with radiolabeled cyclin B2 3′UTR in the presence of 0, 1, 5, or 10 molar excess unlabeled cyclin B2 3′UTR or A1ΔARE. Markers are in kDa. B, RNA-pulldown of biotinylated cyclin B2 3′UTR with GST-ElrA and increasing GST-AUF1 (left panel), or GST-AUF1 and increasing GST-ElrA; followed by immunoblotting for ElrA and AUF1. C, Diagram of cyclin B2 3′UTR deletion mutants. Mutants contain the 30-nt regions shown (1-6) and the NPS (A2UA3). CPE, regions containing this element. D, RNA-pulldown with the biotinylated RNA indicated and 0.05 μg GST-ElrA or 0.2 μg GST-AUF1, followed by immunoblotting for ElrA and AUF1. n=3 for A and B; n=4 for D.

Figure 3

Figure 3. CPEs are not required for ElrA and AUF1 binding

A, UV-crosslink analysis of GST-AUF1 or GST-ElrA with radiolabeled RNA: 1: GbB2, 2-4: GbB2 with region 4 (CPE4-), region 6 (CPE6-) or both CPEs deleted (CPE4-6-). Markers are in kDa. B and C, The indicated RNA was injected into 2-cell embryos, extracted at the indicated hpf (top numbers) and resolved on denaturing gels. U, RNA before injection. n=3.

Figure 4

Figure 4. Overexpression of ElrA and AUF1 does not affect cyclin B2 mRNA stability

2-cell embryos remained uninjected or microinjected with mRNAs encoding myc-tagged AUF1, ElrA, ElrAΔ and processed at the hour indicated (hpf) for either A, myc immunoblotting or B, cyclin B2 Northern analysis. C, Relative amount of cyclin B2 mRNA +/- SEM as compared to 2 hpf uninjected embryos; n=4.

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