Translational control of a subset of mRNAs by GYS1. (A) Cluster analysis on the subset of mRNAs, which were selected for further analyses in Northern blots. The heat-map displays the log2 fold-change. Three classes of mRNAs can be defined: (1) mRNAs with enhanced abundance in heavy polysomes, (2) mRNAs with enhanced abundance in light and medium polysomes, (3) mRNAs with diminished abundance mostly in heavy polysomes. (B) Quantitation of selected mRNAs. Changes in total mRNA abundance of selected mRNAs by Northern blot analysis (red bars), compared to the microarray results (blue bars) of the input sample are shown. (C) Western blot analysis of 4E-BP1, 4E-BP2, GYS1 and actin in cells depleted of or overexpressing GYS1. (D) Quantitation of Western blots for 4E-BP1, 4E-BP2 and GYS1 from independent experiments. The error bars represent the standard error (SE).

Detection of glycogen synthase (GYS1) and phosphorylated GYS1 (pGYS1) in inactive and active ribosomal preparations. (A) Polysomal profiles of preparative gradients from HeLa cell extracts grown in suspension. Cytoplasmic lysates were treated with cycloheximide and separated by ultracentrifugation in 10–50% sucrose gradients. The absorbance profiles measured at 254 nm of collected gradient fractions are shown. The positions of 40S and 60S ribosomal subunits (inactive ribosomes) and polysomes (active ribosomes) are shown. Due to the large scale preparation, the peaks are broadened resulting in the fusion of the 80S with the 60S peak. (B) Inactive 40S/60S subunits fractions, actively translating polysomal samples and total HeLa lysate were separated by SDS-polyacrylamide gel electrophoresis. The presence of pGYS1, phosphorylated on Ser640, and total GYS1 protein in the polysomal fractions was examined by Western blot analysis. The ribosomal proteins S6 (small subunit) and L13a (large subunits) served as controls for the presence of ribosomal subunits. (C) Total HeLa cell lysate and polysomal ribosome samples were treated with calf intestinal phosphatase (CIP) and separated by SDS-polyacrylamide gel electrophoresis. Western blot analysis was used to detect pGYS1 and GYS1 forms.

Polysomal distribution of GYS1 and pGYS1. (A) HeLa cells were treated with 0.1 mg/ml cycloheximide, lysates prepared, separated in a 10–60% sucrose gradient as shown in Figure 1. Distribution of pGYS1, GYS1 and ribosomal proteins S6 and L13a was examined by Western blot analysis. (B) HeLa cells were treated with 2 mM puromycin for 15 min on ice and 10 min at 37°C, extracts prepared and processed as in (A). (C) Quantitation of the distribution of GYS1 and pGYS1 after cycloheximide and puromycin treatment. The set of Western blots shown in (A) and (B) were used for the shown quantitation and are representative for results from several experiments.

Association of pGYS1 with 60S ribosomal subunits. HeLa cells were treated with cycloheximide (A) or puromycin (B) as described in the legend to Figure 2 and cell lysates were separated in 5–25% sucrose gradients. Absorbance at 254 nm was measured. Absorbance peaks that correspond to the 40S and 60S subunits are indicated. The distribution of GYS1, pGYS1, S6 and L13a was analyzed by Western blot. (C) Purified 40S and 60S subunit fractions were separated in SDS-polyacrylamide gels and analyzed in Western blots using antibodies directed against pGYS1, GYS1. S6 and L13a.

Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

Microarray profiling of polysomal fractions isolated from control siRNA- and GYS1 siRNA-treated cells. (A) HeLa cells were transfected with siRNAs and separated on gradients as shown in Figure 4B. Sucrose gradient fractions 8–9, 10–11, and 12–13 were pooled and are indicated as light, medium and heavy polysomes, respectively (see Figure 4B). PolyA-selected RNA was prepared from the gradient input fraction and the various polysomal fractions. Human whole genome oligonucleotide arrays were performed in triplicate on each sample and normalized and averaged on OneArray platforms, performed by Phalanx Biotech. (Palo Alto, CA). Samples from cells transfected with GYS1 siRNAs were compared to samples from cells transfected with control siRNAs. Cluster analysis of genes with a differential p-value of <0.1 was performed. The scale of the heat-map is presented as log2 fold-change. RNAs were categorized into four classes: (1) mRNA whose abundance was diminished in both input and polysomal samples, (2) mRNA whose abundance was diminished in the input sample, but enhanced in heavy polysomal samples, (3) mRNA whose abundance was enhanced in the input sample, but diminished in heavy polysomal fraction, (4) mRNA whose abundance was enhanced in both input and polysomal samples. Several mRNAs displaying significant changes within each of those four classes were chosen for further analysis. (B) Quantitation of selected mRNAs. Changes in total mRNA abundance of selected mRNAs by Northern blot analysis (red bars), compared to the microarray results (blue bars) of the input sample are shown.

Correlation of mRNA abundance and polysomal association. The mRNAs from the heat map in Figure 5 were divided into two groups. Group 1 contains mRNAs with a decreased abundance (log2<0), and group 2 contains mRNAs with an increased abundance (log2>0) in cells depleted of GYS1. The microarray intensities of mRNAs in polysomes, expressed as a fraction mRNA_{polysomal fraction}/mRNA_total in siRNA GYS1-treated samples divided by mRNA_{polysomal fraction}/mRNA_total in siRNA control-treated samples, were divided into three groups. These include mRNAs found to be enriched (log2>0.5) in polysomes, mRNAs found to be depleted in polysomes (log2<−0.5), and mRNAs with an unchanged polysomal association (−0.5<log2<0.5).