Project description:Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict, leaving uncertainty as to the therapeutic benefits of Nutlin. Here, we report a new translation control mechanism shaping p53-dependent apoptosis. We performed polysomal profiling in the cell lines SJSA1 and HCT116, of which only the first undergoes robust cell death in response to p53 activation by Nutlin. We establish Nutlin-induced apoptosis in SJSA1 cells to be associated with a set of translationally enhanced mRNAs carrying a newly identified 3’UTR motif which we labeled CG-motif. We identified PCBP2 and DHX30 as interactor proteins of the CG-motif that are more abundant in HCT116 cells compared to SJSA1. The binding of DHX30 to the CG-motif detected in HCT116 cells was shown to be dependent on PCBP2. Interestingly, DHX30 depletion enhances polysome association of CG-motif mRNAs in HCT116 cells, shifting their outcome towards apoptosis. In U2OS, an osteosarcoma-derived cell line that unlike SJSA1 undergoes persistent cell cycle arrest in response to Nutlin, DHX30 is highly expressed and its depletion enhances the expression of CG-motif mRNAs.
Project description:Recent evidence suggests that non-coding RNAs play an integral regulatory role in numerous functions in lung cancer development. Here, we report identification of a novel lncRNA, herein termed TP53-inhibiting lncRNA (TILR), which plays a role as a constitutive negative regulator of p53 expression and hence its functions including activation of downstream genes such as p21 and MDM2 and induction of apoptosis. Proteomic search for TILR-associated protein revealed the association with PCBP2, and the mid-portion of TILR was required for both PCBP2 and p53 mRNA binding. In addition, depletion of PCBP2 faithfully phenocopied effects of TILR silencing. We also found that TILR suppressed p53 expression post-transcriptionally as well as via a positive feedback loop between p53 and the Fanconi anemia pathway genes. Taken together, our findings clearly demonstrate that TILR constitutively inhibits p53 expression in cooperation with PCBP2, maintaining p53 transcriptional activity at a level sufficiently low for avoidance of spurious apoptosis induction.
Project description:Recent evidence suggests that non-coding RNAs play an integral regulatory role in numerous functions in lung cancer development. Here, we report identification of a novel lncRNA, herein termed TP53-inhibiting lncRNA (TILR), which plays a role as a constitutive negative regulator of p53 expression and hence its functions including activation of downstream genes such as p21 and MDM2 and induction of apoptosis. Proteomic search for TILR-associated protein revealed the association with PCBP2, and the mid-portion of TILR was required for both PCBP2 and p53 mRNA binding. In addition, depletion of PCBP2 faithfully phenocopied effects of TILR silencing. We also found that TILR suppressed p53 expression post-transcriptionally as well as via a positive feedback loop between p53 and the Fanconi anemia pathway genes. Taken together, our findings clearly demonstrate that TILR constitutively inhibits p53 expression in cooperation with PCBP2, maintaining p53 transcriptional activity at a level sufficiently low for avoidance of spurious apoptosis induction.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
