Application): A significant characteristic of cellular senescence is the loss of proliferative capacity, during which cells exhibit morphological changes and alterations in gene expression. Gene expression in eukaryotic cells is regulated to a large extent by post-transcriptional modifications of pre-mRNA, which includes polyadenylation, cleavage, and alternative splicing. Although post- transcriptional process has been investigated in other systems, its relevance in cellular aging has not been examined. This investigator has identified alterations in the levels and activities of proteins that bind to heterogeneous nuclear RNA (hnRNA) in senescent fibroblasts; these are the hnRNP A1 and A2 proteins. Both are functional in the biogenesis and stability of mature mRNA. In addition, increased activities of two novel RNA-binding proteins (LPA-38 and LPA-45) have been uncovered in senescent fibroblasts; these are the hnRNP A1 and A2 proteins. Both are increased activities of two novel RNA-binding proteins (LPA-38 and LPA-45) have been uncovered in senescent fibroblasts. The hypothesis for this study is that alterations in post- transcriptional processing have an important role in affecting gene expression during cellular senescence. The goals of this proposal are to investigate the age-related roles of specific RNA-binding proteins. Changes in post-transcriptional processing in senescent cells will be assessed by using RNA binding and splicing assays. Assessment will be made of whether lifespan will be altered by manipulating the expression of hnRNP A1 and A2 proteins in normal human fibroblasts using an inducible retroviral system. Based on preliminary findings, it is anticipated that lifespan will be lengthened. The effects on lifespan would be the result of hnRNP A1 and A2 modulating the expression of specific RNA species. Subsequently the investigator will seek to identify these RNAs by the use of differential display. Secondly, the genes for LPA-38 and 45, which may be putative negative growth, regulators, will be isolated and characterized. Once their cDNAs have been cloned and sequenced, functional assays will be performed tp determine relevance during cellular senescence. The altered activities of RNA-binding proteins (hnRNP A1 and A2, LPA-38 and 45) are expected to produce several effects for specific RNAs, such as reduced mRNA, defective proteins, and inefficient translation. All are potential mechanisms to induce cellular senescence. The long-term goals of the studies outlined in this application are to characterize the genes identified by differential display and to assess the contribution post- transcriptional processing has in modulating senescence-related gene expression.
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