Slevin MK , Gourronc F , Hartley RS .

CA09589801A1 NCI NIH HHS , CA095898-01A1 NCI NIH HHS , R01 CA095898 NCI NIH HHS

	Figure 1. GST-ElrA binds the 3′UTR of cyclin E1 mRNA. (A) UV-cross-link assay: radiolabeled cyclin E1 3′UTR (E1) was incubated with GST-ElrA, GST or 4 and 8 h embryo extracts. E1 lane is the RNA without added protein or RNase T1. RNase T1 lane is E1 digested with this enzyme in the absence of added protein. Molecular weight markers are indicated to the left of the phosphorimage in kDa. The arrow indicates a 36-kDa protein that binds in embryo extracts. n = 5. (B) Comassie blue stained gel of purified GST-ElrA. Molecular weight markers are indicated to the left of the gel in kDa. (C) Gel shift competition assay: GST-ElrA was incubated with radiolabeled cyclin E1 3′UTR containing a poly(A)65 tail (E1pA) in the presence of increasing molar excess (M) of either unlabeled E1pA or an unlabeled competitor RNA (A1, cyclin A1 3′UTR). E1pA lane is the RNA alone without added protein or RNase T1. Arrow indicates ElrA bound mRNA. Samples were electrophoresed on native gels and analyzed by phosphorimaging; the digested unbound RNA running at the bottom of the gel is not shown. Experiment was also performed in the absence of a poly(A)65 tail with identical results. n = 3. (D) Binding assay to determine the dissociation constant of GST-ElrA for the cyclin E1 3′UTR. Radiolabeled cyclin E1 3′UTR (1 fmol) was incubated with GST-ElrA over a range of 0–50 nM. Reactions were electrophoresed on native gels and analyzed by phosphorimaging. The bracket shows bound RNA and the line shows free RNA (n = 4). The results of the binding assay were quantitated to determine the dissociation constant (Kd,) shown in panel (E).
	Figure 2. Endogenous ElrA binds the 3′UTR of cyclin E1 mRNA. Radiolabeled cyclin E1 3′UTR or cyclin A1 3′UTR was microinjected into two cell-embryos between 1.5 and 2 h post-fertilization (hpf) and time points were taken at 4 and 8 h. Protein extracts were prepared, treated with RNase T1, and UV-cross-linked to preserve RNA–protein complexes. Cross-linked embryo extracts were immunoprecipitated with either anti-HuR, anti-ElrA or pre-immune serum as described in the materials and methods section. Phosphorimages of the immunoprecipitated RNA–protein complexes are shown. Molecular weight markers are indicated to the right in kDa. n = 3.
	Figure 3. ElrA binds the distal portion of the cyclin E1 3′UTR. (A) Schematic of 3′UTR deletions. E1 is the full-length 3′UTR of cyclin E1 mRNA with nucleotides (numbers), restriction sites (E-EcoRI, A-AvaI, EV-EcoRV) and the relative positions of the cis-elements indicated (ARE, AU-rich element; CPE, cytoplasmic polyadenylation element; NPS, nuclear polyadenylation sequence). ▵1575-1784 has the distal part of the 3′UTR deleted while Δ1387-1571 has the proximal part deleted. Phosphorimages of UV-cross-link assays of GST-ElrA with (B) ▵1575-1784 or (C) Δ1387-1571 are shown. The indicated radiolabeled RNA was incubated with GST-ElrA, GST or 4- and 8-h embryo extracts. RNase T1 lane is the RNA digested with this enzyme in the absence of added protein. Molecular weight markers are indicated to the left in kDa. The arrow indicates a 36-kDa protein that binds in embryo extracts. n = 3.
	Figure 4. Cyclin E1 3′UTR structure as predicted by the mfold algorithm (22). The entire 3′UTR of either X. tropicalis (A, Genbank accession NM_001016328) or X. laevis (B, Genbank accession Z13966) was folded using the mfold algorithm. The most stable predicted structure is shown, along with its free-energy value (dG) as compared to the initial free energy before folding. Only the distal part of the 3′UTR is shown due to space limitations (X. laevis nt 1641–1784, X. tropicalis nt 1617–1861). Large numbers show similar sequences and/or structures between the species as explained in the text. Small numbers refer to nucleotides with the first nucleotide of the 3′UTR set as 1.
	Figure 5. The distal portion of the 3′UTR of cyclin E1 mRNA specifies polyadenylation. Radiolabeled cyclin E1 3′UTR (A) or 3′UTR deletions (B) ▵1575-1784 or (C) Δ1387-1571 were injected into two cell-embryos. Total RNA was extracted at the indicated hour post-fertilization (hpf), electrophoresed on a denaturing gel and analyzed by phosphorimaging. U is RNA before injection. Nucleotide markers are indicated on the left. Bracket indicates polyadenylated RNA. The 2-hpf sample was taken 5 min post-injection. n = 5.
	Figure 6. Polyadenylation requires a minimum of three cis-elements in the 3′UTR. (A) Schematic of 3′UTR deletion mutants used in panels (B–G). The names of the RNAs and the nucleotides deleted are indicated on the right. E1 is the full-length cyclin E1 3′UTR as described in Figure 3. (B–G) show polyadenylation assays for the 3′UTR deletion mutants indicated performed as described in Figure 3. U is RNA before injection. Nucleotide markers are indicated on the left. Bracket indicates polyadenylated RNA. The 2-hpf sample was taken 5-min post-injection. n = 2 for panel E; n = 3 for panels C, D and F; n = 4 for panels A and G. (H) Graph showing the percent of indicated RNA remaining (+/− SEM) at each timepoint averaged over all experiments. Quantitation was performed with Imagequant (Molecular Dynamics).
	Figure 7. Deletion of the 3′UTR cis-elements disrupts ElrA binding. Here, 1 fmol of the indicated radiolabeled RNA was incubated with GST-ElrA over a range of 0–50 nM. Reactions were electrophoresed on native gels, phosphorimaged (top panels) and the results quantitated (bottom panels). Bound and free RNA are indicated by the lines on the right. n = 2 for panels (A and B), n = 3 for panel (C).

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