Gene regulation by post-transcriptional processing during cellular senescence in human diploid fibroblasts.

摘要

Primary human fibroblasts undergo an extended period of senescence at the end of their replicative life span which is characterized by the complete loss of proliferative potential accompanied by specific morphological and molecular changes. The expression of a gene from the time it is transcribed to the moment a functional protein is made can be modulated at various steps and alternative splicing of pre-mRNA, mRNA transport and regulation of mRNA half-life are important steps that ultimately regulate the levels of functional protein that a cell expresses. We are interested in a family of proteins called Heterogeneous Nuclear Ribonucleoproteins, specifically type A1 and A2 that appear to have roles in many steps of mRNA biogenesis mentioned above. We have identified that levels of these proteins are significantly changed in senescent fibroblasts. However there is very little information regarding their function in senescent cells. My proposed hypothesis was that the observed changes in levels of hnRNP A1 and A2 could have significant effects on the gene expression patterns of senescent cells where their levels are significantly lower and that identifying these changes could help elucidate the mechanism by which cells age.; In order to study altered gene expression patterns of cell cycle regulatory proteins, we performed quantitative RT-PCR and immunoblotting to study specific target genes and products. To identify gene expression changes on a more global scale we performed cDNA array experiments and compared expression profiles among senescent cell populations and fibroblasts that over-express hnRNP A1. We have identified cyclin D1 as a candidate mRNA that may be regulated by age and post-transcriptional processing. In order to identify target mRNA that are bound by hnRNP A1 and maybe age-regulated, we performed RNA co-immunoprecipitation experiments using hnRNP A1 as the bait protein. With this approach we identified human mdm2 mRNA as a binding target for hnRNP A1 in young and old fibroblasts. This is a novel and important finding because a large number of mRNA isoforms are made from the human mdm2 pre-mRNA. Despite its importance in regulation of p53, little is known about the role of the alternatively spliced isoforms of human mdm2.