The focus of this study is to determine the functional role of post- transcriptional processing in cellular senescence. We will use specific monoclonal antibodies to determine the presence and protein levels of hnRNP proteins in extracts prepared from HS74, a human fetal diploid fibroblast cell line. Whole cell and nuclear extracts will be made at intervals representative of its life span. Extracts will also be prepared from SV40-transformed immortalized HS74 fibroblasts and assayed to serve as controls for the age-related alterations in the levels and activities of RNA binding proteins. The significance of post-transcriptional processing will be analyzed employing several in vitro assays. Cleavage and polyadenylation will be monitored in nuclear extracts using a model RNA polyadenylation substrate containing functionally important sequences. Alternative splicing assays will be performed using a fibronectin RNA substrate to provide assessment of this form of processing in a natural sequence context. In addition, we will further characterize the sequence requirements for RNA binding of the two novel RNA binding proteins. These studies will provide a framework to investigate the role of specific RNA binding proteins in cellular senescence. They will also facilitate my long term research plan, to explore the contribution of post- transcriptional processing to altered gene expression observed during cellular aging. Human diploid fibroblasts (HP) have a characteristic limited life span in culture. The terminal post-mitotic phase (cellular senescence) has been extensively used as an in vitro model for aging. Alterations in morphology and numerous differences in the levels of gene expression have generated several theories which attempt to define the loss of proliferative capacity. We have found that the activities and levels of several RNA binding proteins which modulate post-transcriptional processing of pre-mRNA are compromised in senescent cells. The proteins effected in senescence bind to heterogenous nuclear RNA (hnRNA). Many hnRNP proteins participate in the biogenesis' of mature mRNA. The hypothesis of this study is that the senescent phenotype and its subsequent deregulation of proliferation is a result of altered gene expression by modulation of RNA transcripts which are under the influence of the RNA binding proteins.
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