The p27 protein, a key regulator of cell cycle progression, is regulated both transcriptionally and posttranscriptionally (17
). Two posttranscriptional steps, translation and protein degradation, are mainly responsible for the oscillation of p27 levels during cell cycle and the induction of p27 in growth-arrested cells (1
). According to recent reports, increased protein synthesis (rather than proteolytic degradation) plays a major role in determining p27 levels during TPA-induced differentiation of HL60 cells, HeLa cell growth arrest at G1
phase, and contact inhibition of human diploid fibroblasts (15
It has been shown that an IRES element in the 5′NTR of p27 mRNA directs translation of the mRNA (28
). Therefore, it is likely that the IRES activity of the p27 mRNA varies in response to intracellular and extracellular changes that modulate the availability and/or activity of ITAFs. In this respect, IRES-binding proteins, such as HuR, HuD, and hnRNP C1/C2, which interact with the IRES element of the p27 mRNA (28
), are possible candidates for regulation of the p27 IRES. In fact, HuR and the related HuD protein were shown to repress IRES-dependent translation of the p27 mRNA through an interaction with a U-rich element in the IRES element (28
). However, no previous work has identified an ITAF capable of stimulating the IRES element of the p27 mRNA.
Here, we show that PTB enhances p27 IRES activity through an RNA-protein interaction. Several lines of evidence indicate that PTB augments p27 IRES function. First, PTB specifically interacts with the p27 IRES, as shown by UV cross-linking of HeLa cell extracts with 32P-labeled p27 IRES RNA followed by immunoprecipitation with an anti-PTB antibody (Fig. ), and further supported by UV cross-linking of purified PTB proteins with 32P-labeled p27 IRES RNAs in the presence or absence of various cold competitor RNAs (Fig. ). Second, addition of PTB to an in vitro translation reaction mixture increased the activity of a reporter under the control of the p27 IRES element. The effect of PTB on the p27 IRES was higher than that on the EMCV IRES, a positive-control IRES known to use PTB as an ITAF (Fig. ). Third, overexpression of PTB increased the activity of a reporter under the control of the p27 IRES element in an artificial dicistronic mRNA in vivo (Fig. ). Fourth, siRNA-mediated knockdown of PTB levels inhibited the IRES activity of p27, as shown by transfection of DNA (Fig. ) and RNA (Fig. ) reporters. And fifth, the level of p27 protein (which reflect the IRES activity of endogenous p27 mRNA) correlated well with the level of PTB protein during TPA-induced differentiation of HL60 cells (Fig. ).
It is not clear how PTB enhances translation of the p27 mRNA, but we can speculate several modes of action. First, a putative PTB-ribosome interaction may assist in binding or guiding ribosomes to the IRES. Alternatively, PTB may conformationally change the IRES into a structure that is better able to interact with the translational machinery, including the 40S ribosomal subunit. In support of this possibility, a previous study suggested that the RNA chaperone activity of PTB may favor rearrangement of the IRES structure to one that permits translation initiation (34
). Second, it is possible that PTB recruits a canonical translation factor(s) through a putative protein-protein interaction. Third, PTB may activate IRES-dependent translation through interactions with other ITAFs. In this respect, it is noteworthy that many ITAFs are known to interact with each other (22
Cells undergoing differentiation exhibit extensive changes in their patterns of gene expression and a profound reduction of global protein synthesis, as indicated by the release of most mRNAs from polysomes early during TPA-induced differentiation of HL60 cells (27
). However, protein expression is activated in selective genes, as indicated by retention or even migration of a subset of transcripts onto polysomes during differentiation (27
). The association of the p27 mRNA with polysomes is enhanced by TPA treatment of HL60 cells (32
). Unexpectedly, the protein levels of HuR, which was shown to repress the activity of the p27 IRES (28
), gradually increased following TPA treatment of HL60 cells, beginning at 3 h posttreatment and reaching maximal levels at 36 h posttreatment, when the p27 IRES activity was also maximal (Fig. ). This result strongly suggests that the increased IRES activity of the p27 mRNA is not associated with decreases in HuR protein levels during TPA-induced differentiation of HL60 cells. Instead, the activation of the p27 IRES function during differentiation is more likely due to the dramatic increase in PTB protein levels noted 24 h after TPA treatment (Fig. , panel PTB). Interestingly PTB expression also seems to be regulated at the posttranscriptional level, since the level of PTB mRNA remained consistent during TPA-induced differentiation of HL60 cells, as indicated by Northern blot analysis (Fig. , panel PTB mRNA). However, the molecular basis of posttranscriptional regulation of the PTB gene remains to be elucidated.
A few lines of evidence suggest that PTB functions in differentiation and cell cycle control. For instance, loss of Drosophila
PTB specifically affects spermatid differentiation of Drosophila melanogaster
), and PTB-like protein, a rat homolog of PTB, was shown to modulate the differentiation of PC12 cells into neuronal cells (16
). However, this is the first report suggesting that PTB plays a role in cell cycle arrest during cellular differentiation. In this respect, it is noteworthy that the knockdown of PTB by specific siRNAs reduced the proportion of cells in G1
phase relative to those in G2
/M and S phases (Fig. ). Conversely, TPA-induced differentiation of HL60 cells, which causes induction of PTB protein, increased the proportion of cells in G1
phase relative to those in the G2
/M and S phases (Fig. ). This result strongly suggests that the arrest of cell proliferation during TPA-induced differentiation of HL60 cells is most likely due at least in part to enhancement of p27 IRES activity via induction of PTB levels. However, the function of PTB should be further investigated to confirm its precise role during differentiation.
Some IRESs and ITAFs have been reported to regulate the translation of cellular mRNAs that play critical roles in cell cycle progression and differentiation. For example, the platelet-derived growth factor B (PDGF2/c-sis
) mRNA contains an IRES element that shows increased activity during megakaryocytic differentiation of K562 cells (3
). Sella et al. demonstrated that differentiation-dependent activation of the PDGF2/c-sis
IRES correlated with binding of hnRNP C (47
), and Koloteva-Levine et al. showed that Apc5, a component of anaphase-promoting complex/cyclosome (APC/C), inhibited the IRES activity of the PDGF2/c-sis
mRNA in a differentiation-dependent manner (25
). The IRES activity of the c-myc
proto-oncogene, a key regulator of cell proliferation and apoptosis, is regulated during differentiation of a transgenic mouse (9
), and HnRNP C was shown to enhance the activity of the c-myc
IRES in a cell cycle phase-dependent manner (23
). Here, we report that PTB enhances p27 IRES activity. Our data and analysis suggest that IRESs and ITAFs play important roles in fine-tuning the levels of pivotal regulatory proteins during cell proliferation and differentiation. Therefore, this work and future investigations into IRESs and ITAFs will increase our understanding of the molecular basis of cell differentiation and may contribute to the development of novel therapeutic strategies against cancer.