Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest

Abstract

The alpha-globin poly(C)-binding proteins (alphaCPs) comprise an abundant and widely expressed set of K-homolog domain RNA-binding proteins. alphaCPs regulate the expression of a number of cellular and viral mRNAs at the levels of splicing, stability, and translation. Previous surveys have identified 160 mRNAs that are bound by alphaCP in the human hematopoietic cell line, K562. To explore the functions of these alphaCP/mRNA interactions, we identified mRNAs whose levels are altered in K562 cells acutely depleted of the two major alphaCP proteins, alphaCP1 and alphaCP2. Microarray analysis identified 27 mRNAs that are down-regulated and 14 mRNAs that are up-regulated in the alphaCP1/2-co-depleted cells. This alphaCP1/2 co-depletion was also noted to inhibit cell proliferation and trigger a G(1) cell cycle arrest. Targeted analysis of genes involved in cell cycle control revealed a marked increase in p21(WAF) mRNA and protein. Analysis of mRNP complexes in K562 cells demonstrates in vivo association of p21(WAF) mRNA with alphaCP1 and alphaCP2. In vitro binding assays indicate that a 127-nucleotide region of the 3'-untranslated region of p21(WAF) interacts with both alphaCP1 and alphaCP2, and co-depletion of alphaCP1/2 results in a marked increase in p21(WAF) mRNA half-life. p21(WAF) induction and G(1) arrest in the alphaCP1/2-co-depleted cells occur in the absence of p53 and are not observed in cells depleted of the individual alphaCP isoforms. The apparent redundancy in the actions of alphaCP1 and alphaCP2 upon p21(WAF) expression correlates with a parallel redundancy in their effects on cell cycle control. These data reveal a pivotal role for alphaCP1 and alphaCP2 in a p53-independent pathway of p21(WAF) control and cell cycle progression.

FIGURE 1.

siRNA-mediated depletion of αCP1 and αCP2. A, cells were mock-transfected (M) or transfected with siRNAs directed against αCP1, αCP2, αCP1 and αCP2 (αCP1 + 2), or lamin A/C (Lam). Protein lysates were analyzed by Western blotting using antibodies directed against the corresponding proteins. The two bands detected with the αCP2-specific antibodies represent αCP2 full-length protein (upper band) and αCP2KL, its major splice variant. The two bands in the lamin A/C Western blot panel represent the two lamins, A and C. B, the simultaneous depletion of αCP1 and αCP2 results in decreased cyclin H protein expression. Cells were mock-transfected or transfected with a mixture of siRNAs directed againstαCP1 andαCP2 (αCP1 + 2) or directed against lamin A/C (Lam). Protein lysates were analyzed by Western blotting using antibodies directed against cyclin H or an antibody that recognizes RB (as a loading control).

FIGURE 2.

K562 cells that are acutely co-depleted of αCP1 and αCP2 accumulate in the G₁ phase of the cell cycle. A, cells were mock-transfected or transfected with siRNAs directed against αCP1, αCP2, αCP1 and αCP2 (αCP1 + 2), or lamin A/C (Lam) and were subject to cell cycle analysis at 4 or 5 days after siRNA treatment. The x axis shows DNA content as determined by propidium iodide (PI) fluorescence, and the y axis shows number of cells. The regions representing G₁, S, and G₂ are indicated. B, analysis and quantitation of the percentage of cells found in each phase of the cell cycle from replicate experiments. The mean and S.D. are shown.

FIGURE 3.

Co-depletion of αCP1 and αCP2 increases the phosphorylation of RB at serine 795. Cells were mock-transfected (M) or transfected with siRNAs directed against αCP1 and αCP2 (αCP1 + 2) or lamin A/C (Lam). Protein lysates were analyzed by Western blotting using antibodies directed against the following phosphorylated forms of RB: Ser⁷⁹⁵ and Ser⁷⁸⁰ (A) and Ser^807/811 (B). An antibody that recognizes RB was utilized as a load control.

FIGURE 4.

Co-depletion ofαCP1 andαCP2 results in p53-independent induction of p21^WAF expression. A, K562 cells were mock-transfected (M) or transfected with siRNAs directed against αCP1, αCP2, αCP1 and αCP2 (αCP1 + 2), or lamin A/C (Lam). HCT116 cells that lack (–/–) or contain the wild type (WT) p53 gene were treated with irinotecan to induce p53 expression and were used as controls. Protein lysates were analyzed by Western blotting using antibodies directed against p53. The loading control is the ribosomal protein L7 (rpL7). B, K562 cells were transfected with siRNAs as above and analyzed by Western blotting using antibodies directed against p21^WAF. An antibody directed against RB was utilized as a loading control.

FIGURE 5.

The mRNA encoding p21^WAF is associated with αCP1 and αCP2 in vivo. K562 cell extracts were immunoprecipitated (IP) with antibodies to αCP1 or αCP2 or a c-Myc control (Cont) antibody. Following immunoprecipitation, RNA was isolated from the RNP complexes and subjected to RT-PCR analysis to detect p21^WAF or γ-globin mRNAs. The γ-globin image for the αCP1 and control immunoprecipitate was from different areas of the same gel.

FIGURE 6.

The p21^WAF mRNA is stabilized in cells co-depleted of αCP1 and αCP2. A, K562 cells were mock-transfected (M) or transfected with siRNAs directed against αCP1 and αCP2 (αCP1 + 2) or against lamin A/C (Lam). At 2 days post-transfection, actinomycin D was added to inhibit transcription, and total RNA was collected at 0, 2, and 4 h. Northern blot analysis was used to monitor p21^WAF mRNA levels. Representative blots are shown. B, the experiment shown in A was performed three times, and the p21^WAF mRNA levels at each time point were quantified on a PhosphorImager. For each set of siRNA transfections, the band intensity at the 0 h time point was set to 100%, and the percentage of mRNA remaining was plotted over time. The error bars show the S.E. among the three experiments.

FIGURE 7.

αCP1 and αCP2 bind to a 127-nucleotide fragment of the p21^WAF 3′-UTR. A, fragments of the p21^WAF 3′-UTR used in the initial cross-linking assay (33) and sequences corresponding to subfragments of WAF 1–879 used in higher resolution mapping. B, UV cross-linking assay of fragments of the p21^WAF 3′-UTR. Cytoplasmic extracts from K562 cells were incubated with thiolated, ³²P-labeled RNA sequences representing different regions of the p21^WAF 3′-UTR. The mixture was cross-linked, digested with RNase, and immunoprecipitated (IP) with antibodies directed against c-Myc (Control), αCP1, or αCP2. The resulting complexes were analyzed by SDS-PAGE. Molecular weight markers are shown on the right. The top panel shows the cross-linking results with the RNAs described in the first set of fragments shown in A. An actin antisense RNA (Actin AS) was utilized as a negative control, and the α-globin 3′-UTR (α-globin) was utilized as a positive control. The bottom panel shows the cross-linking results with the RNAs shown in the second set of fragments in A. The WAF 1–879 was included as a positive control in this second study. C, nucleotide sequence of WAF1-A. Three CU-rich patches are underlined.