Abstract
Post-transcriptional control of gene expression is mediated by the interaction of RNA-binding proteins with their cognate mRNAs that specifically regulate their stability, localization and translation. mRNA-binding proteins are multifunctional and it has been proposed therefore that a combinatorial RNA-binding protein code exists that allows specific protein sub-complexes to control cytoplasmic gene expression under a range of pathophysiological conditions. We show that polypyrimidine tract-binding protein (PTB) is central to one such complex that forms in apoptotic cells. Thus, during apoptosis initiated by TNF-related apoptosis inducing ligand there is a change in the repertoire of RNA-binding proteins with which PTB interacts. We show that altering the cellular levels of PTB and its binding partners, either singly or in combination, is sufficient to directly change the rates of apoptosis with increased expression of PTB, YBX1, PSF and NONO/p54nrb accelerating this process. Mechanistically, we show that these proteins post-transcriptionally regulate gene expression, and therefore apoptotic rates, by interacting with and stimulating the activity of RNA elements (internal ribosome entry segments) found in mRNAs that are translated during apoptosis. Taken together, our data show that PTB function is controlled by a set of co-recruited proteins and importantly provide further evidence that it is possible to dictate cell fate by modulating cytoplasmic gene expression pathways alone.
Highlights
During pathophysiological conditions of stress including viral infection, amino-acid starvation and apoptosis cells respond by reprogramming protein synthesis.[1]
MCF7 cells were exposed to TNF-related apoptosis inducing ligand (TRAIL) to initiate apoptosis, and nuclear and cytoplasmic extracts were generated from either apoptotic or control cells
Samples were subjected to tandem LC-MS/MS mass spectrometric analysis; this analysis was repeated on a further two independent occasions. Both the number of unique peptides identified for PTB and the immunoblot analysis show that there was an increase in the amount of PTB in the cytoplasm during TRAIL treatment, and a corresponding decrease in the amount of PTB in the nucleus (Figure 1ai), in agreement with previous data.[2,14]
Summary
During pathophysiological conditions of stress including viral infection, amino-acid starvation and apoptosis cells respond by reprogramming protein synthesis.[1]. The inhibition of protein synthesis that occurs during cell stress is achieved by modification of the canonical protein synthesis machinery (eukaryotic initiation factors) and cells have developed specialized mechanisms to bring about the recruitment of selective subsets of mRNAs to actively translate ribosomes under these conditions This is mediated by the interactions of RNA elements in the 50 and 30 UTRs of mRNAs with their cognate mRNAbinding proteins/regulatory RNAs.[3] Many cis-acting mRNA regulatory elements that permit selective translation under different conditions of cell stress have been identified including internal ribosome entry segments (IRESs)[2] and upstream open reading frames[4] in the 50 UTR and miRNAbinding sites and stability determinants[3] in the 30 UTR. PTB is found in large ribonuclear complexes and several known binding partners have been identified, for example, PSF and hnRNPL.[11,12,13] the IRESs that function during TRAIL-mediated apoptosis are PTB dependent and we Received 07.2.13; revised 29.8.13; accepted 30.8.13; Edited by RA Knight; published online 18.10.13
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