Fig. 1. RKO cells expressing reduced HuR show reduced growth rates. (A) Western blot analysis depicting HuR and AUF1 expression in RKO cells transfected with the empty pSVzeo vector (zeo), and in two RKO clonal lines transfected with pSVzeo(–) HuR to express an antisense HuR transcript (AS.17 and AS.11). Blots were sequentially stripped and rehybridized to assess the expression of AUF1 and actin. (B) Proliferation rates in RKO zeo, AS.17 and AS.11 cells, measured after seeding 10 000 cells and counting at 24 h intervals using a hemacytometer. (C) Representative FACS histograms of RKO zeo, AS.17 and AS.11 cultures undergoing logarithmic growth. Data represent the mean ± SEM of four independent experiments.

Fig. 3. Cyclin mRNA expression and stability during the cell division cycle. (A) Left: representative Northern blot analysis of the expression of cyclins D1, E, A and B1. After a 3 day synchronization period, cells were serum stimulated for the times shown, whereupon RNA was extracted for analysis. Blots were sequentially stripped and rehybridized to detect each of the transcripts shown; 18S rRNA signals served to quantitate differences in loading and transfer among samples. Right: quantitation of the levels of each cyclin mRNA from three independent experiments. Data are represented as fold induction in mRNA levels relative to those at time 0 (before serum addition). (B) The half-lives of cyclin A and cyclin B1 mRNAs in either asynchronous (asyn.) or synchronous populations (serum stimulated for the times indicated) were calculated after adding 2 µg/ml actinomycin D after each serum stimulation period, preparing RNA at the times indicated thereafter, measuring the remaining signals of cyclin A and cyclin B1 mRNAs by Northern blot analysis, normalizing them to 18S rRNA, and plotting them on a logarithmic scale (inset). mRNA half-lives at each time following serum stimulation are indicated in parentheses.

Fig. 5. Cell cycle-dependent binding to transcripts encoding cyclins A and cyclin B1. Binding activity of radiolabeled RNAs corresponding to either the coding regions or 3′ UTRs of cyclins D1, E, A and B1, and proteins present in cytoplasmic lysates of asynchronous (asyn.) or synchronous RKO cultures after addition of serum for the times indicated is shown.

Fig. 7. Binding of RKO ASHuR cells to transcripts from cyclins A and B1. (A) Quantitation of radioactive signals (sum of all bands, represented as the mean ± SEM from three independent experiments) from binding experiments similar to those shown in (B). (B) Repre sentative complexes forming between cytoplasmic lysates from synchronous populations of RKO zeo or ASHuR cells (prepared at the times indicated after serum stimulation), and radiolabeled cyclin A 3′ UTR (top) or cyclin B1 3′ UTR (bottom). Supershift analysis was carried out where indicated (+HuR); the arrowhead shows supershifted complexes.

Fig. 9. Cyclin A and B1 protein levels and cdk2- and cdc2-associated kinase activity in ASHuR cells. (A) Representative Western blots of cyclin A and cyclin B1 in whole-cell lysates prepared from synchronous RKO zeo and AS.11 cells at the times indicated after serum stimulation. Actin signals served to monitor the equality of sample loading and transfer. (B) Representative cdk2 and cdc2 kinase activities in RKO zeo and ASHuR cells after serum stimulation for the times indicated. Phosphorylated histone H1 is shown.

Fig. 2. Cell cycle-dependent cytoplasmic localization of HuR. (A) Thymidine incorporation in synchronous RKO zeo populations after serum addition. Data represent the mean ± SEM of four independent experiments. (B) Western blot analysis of HuR (34 kDa) levels in the cytoplasmic (Cyto.) and nuclear (Nuc.) fractions of RKO cells that were either serum starved for 3 days, then serum stimulated for the times indicated, or asynchronously growing (Asyn.). To monitor the quality of the fractionation procedure and the evenness in loading and transfer among samples, membranes were stripped and rehybridized to detect either actin (cytoplasmic, 43 kDa) or BAF57c (nuclear, 57 kDa). (C) Western blot analysis of HuR levels in the cytoplasmic and nuclear fractions of synchronous mouse embryo fibroblast populations that were either serum starved for 3 days, then stimulated by addition of 10% serum, or grown to high density (high), then replated at low density.

Fig. 4. HuR binds transcripts encoding cyclins A, B1 and D1. (A) Schematic representation of mRNAs encoding cyclins D1, E, A and B1. Small circles represent AUUUA elements. (B) RNase T1 selection assay. Radiolabeled RNAs were incubated in the presence (+) or absence (–) of 10 nM GST–HuR, then digested with RNase T1. (C) Nitrocellulose filter binding assays were performed after incubating radiolabeled transcripts with the indicated concentrations of GST–HuR; percentages of bound RNA are shown.

Fig. 6. HuR forms complexes with cyclin A and cyclin B1 3′ UTR transcripts. (A) Complexes forming with lysates from synchronous RKO zeo cells (18 h serum stimulated) and radiolabeled 3′ UTR transcripts from cyclins A and B1 were assayed for their ability to be supershifted by the indicated antibodies. (B) Biotinylated transcripts (sense, 3′ UTR; antisense, complementary to each 3′ UTR) corresponding to the 3′ UTRs of either cyclin A or cyclin B1 were incubated with cytoplasmic RKO lysates (prepared 0 or 18 h after serum stimulation). Complexes were pulled down using streptavidin-conjugated magnetic beads and analyzed by Western blotting to assess HuR levels. lys., 35 µg of RKO zeo whole-cell lysate.

Fig. 8. Stability and levels of mRNAs encoding cyclins A and B1. (A) Half-lives of mRNAs encoding cyclins A and B1 in AS.11 populations were calculated as described in Figure 3B. mRNA half-lives at each time point following serum stimulation are indicated in parentheses. (B) Relative steady-state levels of mRNAs encoding cyclins A and B1 in RKO untransfected (untr.), zeo, AS.17 and AS.11 cells. Data represent the mean ± SEM from eight independent Northern blots.