The effects of poly (A)-binding protein 1 (PABP1) over-expression on mRNA metabolism and the regulation of PABP1 expression following recovery from heat shock
Cytoplasmic poly (A)-binding protein (PABP1) plays crucial roles in mRNA translation and stability, and its expression is tightly regulated. Previous studies found the cellular level of 19 mRNAs was reduced in PABP1 over-expressing cells. This thesis explores some of the mechanistic relationships between PABP1 over-expression and the regulation of specific mRNAs, and also the regulation of PABP1 expression under heat shock. One objective examined the mechanism by which PABP1 expression reduced the level of mitogen-activated protein kinase kinase 2 (MKK2) mRNA. This particular gene was chosen because it functions in the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, and its perturbation is implicated in cellular transformation and human cancer. The specific hypothesis being tested is that PABP1 binding to a specific region of the MKK2 mRNA induces degradation of the transcript. This hypothesis was examined by UV cross-linking of PABP1 and MKK2 mRNA. This approach revealed a novel stability control element in the 3' untranslated region of the MKK2 mRNA that binds PABP1. The control element decreases the stability of a reporter mRNA in PABP1 over-expressing cells. Furthermore, it was also determined that PABP1 was hypo-phosphorylated in PABP1 over-expressing cells, and that the MKK2-ERK1/2 kinase pathway was involved in the phosphorylation of PABP1. A second objective investigated the role and status of PABP1 in response to heat shock because PABP1 is important in the regulation of mRNA translation and cap-dependent mRNA translation is inhibited during heat shock. The hypothesis is that if PABP1 is involved in the regulation of gene expression during and after heat shock, then the interaction of PABP1 with eukaryotic initiation factor 4G (eIF4G) may change. Therefore, the interaction of PABP1 with eIF4G and the regulation of PABP1 expression following heat shock and recovery were examined. Under heat shock in HeLa cells, PABP1 and elF4G became insoluble granules and were translocated from the cytoplasm into the nucleus. PABP1 dissociated from eIF4G and localized in nuclear granules. PABP1 co-localized with heat shock protein 70 (HSP70), whereas eIF4G co-localized with HSP27. Moreover, we found that the abundance of PABP1 was up-regulated during recovery from heat shock at the level of mRNA translation by the 5' Terminal Oligo Pyrimidine (TOP) 'cis'-element located at the 5' UTR of PABP1 mRNA. Keywords: cytoplasmic poly (A)-binding protein (PABP1), mitogen-activated protein kinase kinase 2(MKK2), mRNA stability, heat shock, eukaryotic translation initiation factor (elF)G6, translational control heat shock protein 27 (HSP27), HSP70.