Regulation of c-myc mRNA Decay in Vitro by a Phorbol Ester-inducible, Ribosome-associated Component in Differentiating Megakaryoblasts

Abstract

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation. TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells. A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution. One component fractionates to the postribosomal supernatant from either untreated or treated cells. This component is sensitive to cycloheximide and micrococcal nuclease. The other component is polysome-associated and is induced or activated by TPA. Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway. The cell-free mRNA decay system reconstitutes this alternate decay pathway as well.

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

“…The RNAsedependent decay of c-myc mRNA is one of the critical checkpoints in the process of erythropoetic differentiation, because cells must negate the proliferating activity of c-myc before the onset of differentiation. 6,12,14 Our results showed an increased specificity of proteasomal RNAse towards the c-myc 3'UTR in differentiated K562 cells, suggesting that overexpression of 26S proteasome may also destabilize c-myc mRNA in vivo during hemin-induced differentiation.…”

“…Accordingly, stability of the c-myc mRNA is regulated via ARE-mediated decay during differentiation of proerythroleukaemic K562 cells to megakaryoblasts. 14 Although transcription of the c-myc gene appears to be constitutive during differentiation, the steady-state level of c-myc mRNA declines at least 10-fold. 14 In addition, c-myc is controlled by proteasome at the protein level, which adds another layer of complexity to regulation of its expression.…”

“…14 Although transcription of the c-myc gene appears to be constitutive during differentiation, the steady-state level of c-myc mRNA declines at least 10-fold. 14 In addition, c-myc is controlled by proteasome at the protein level, which adds another layer of complexity to regulation of its expression. 15 The 26S proteasome is post-translationally modified by phosphorylation as well as by N-acetylation, glycosylation, 4-hydroxy-2-nonenal-alkylation in different species.…”

Targeted Knockdown of the RNA-binding Protein CRD-BP Promotes Cell Proliferation via an Insulin-like Growth Factor II-dependent Pathway in Human K562 Leukemia Cells

“…The c-myc antisense probe yields multiple protected fragments corresponding to mRNA molecules with closely spaced polyadenylation sites (1). ␥-Globin mRNA served as the internal control.…”

“…RNase Protection Assay-c-myc and ␥-globin mRNA levels were examined by RNase protection assay in a single tube format as described previously (1). The c-myc antisense probe yields multiple protected fragments corresponding to mRNA molecules with closely spaced polyadenylation sites (1).…”