Figure 1.
Pathway analysis of genes differentially expressed in Pin1 siRNA treated cells vs. controls.
(A) Western blot showing the decrease in Pin1 protein levels in Pin1 siRNA treated cells used for microarray analysis. Actin is used as the loading control. (B) Functional clustering of genes based upon the major biological pathways targeted in Pin1 knockdown cells is shown. Pathway-Express software was used to identify pathways most affected by Pin1 silencing. The graph represent pathways ranked according to impact factor (in parentheses), a measure of the degree of pathway perturbation.
Figure 2.
The mRNA abundance of histone mRNAs and ARE-containing mRNAs is altered in response to Pin1 silencing.
Heat maps of histone mRNAs (A) and ARE-containing mRNAs (B and C) whose expression was significantly altered in Pin1 siRNA treated cells relative to control cells in the microarray data analysis. The heat maps depict the mean fold-change (F.C) for genes that had a p value <0.2. Panel B shows ARE containing genes that were up regulated in Pin1 siRNA treated cells, panel C shows ARE containing genes down-regulated in Pin1 siRNA treated cells. (D–F) The mRNA levels of a subset of genes that were significantly up or down-regulated in the microarray analysis were analyzed by RT-PCR as described in Methods. In (D), the fold change determined by RT-PCR for a subset of histone mRNAs is shown. Fig. D has been reproduced from (Fig. 7 panel A) Krishnan et al MCB (2012) 32:21, 4306–4322. In (E), data was validated by RT-PCR for a subset of genes that were up-regulated in Pin1 siRNA treated cells, whereas (F) shows data for genes that were down-regulated. Genes for which there was good agreement by both microarray and RT-PCR data are shown as filled bars.
Figure 3.
Pin1 interacts with a subset of phosphorylated RNA binding proteins.
Cell lysates from 293T cells were immunoprecipitated with either an anti-Pin1 or anti-hemagglutinin (HA)-specific antibody. The immunoprecipitates were resolved on a 15% SDS-PAGE and analyzed by western blotting for several ARE-BPs, Pin1, and SLBP. Five percent of the input sample was analyzed in lane 1, proteins bound to the anti-Pin1 antibody in the presence (lane 2) or absence of RNAseA (lane 3), and proteins bound to the anti-HA antibody in the absence of RNAseA in lane 4.
Figure 4.
Pin1 acts in concert with specific RNA binding proteins to regulate the mRNA abundance of histone mRNAs and the c-FOS mRNA.
(A) qRT-PCR analysis of histone mRNA expression in response to siRNA knockdown of Pin1, SLBP, and a double Pin1/SLBP RNAi knockdown in HEK293T cells is shown for a subset of histone mRNAs. The average change in histone mRNA levels for all five histone genes probed is shown in (B). In (C), qRT-PCR analysis of the c-FOS mRNA in response to siRNA knockdown of Pin1, HuR, AUF1, KSRP, and the double Pin1/ARE-BP RNAi knockdown in HeLa cells is shown.
Figure 5.
Correlation between mRNA half-lives of the target genes identified and change in mRNA abundance in the Pin1 siRNA knockdown.
(Top) In (A), the fold change in mRNA abundance for genes whose mRNA levels increase at least two fold is plotted against their half-life. In (B), the fold change in mRNA abundance for genes whose mRNA levels decrease at least two fold is plotted against their half-life. (Bottom) Frequency distribution of target genes that are either stabilized (left) or destabilized (right) in Pin1 siRNA treated cells as a function of mRNA half-life. Genes that show the largest fold stabilization in a Pin1 siRNA knockdown have half-lives <4 hr.