Influence of mRNA Turnover on the Biology of Cellular Senescence and Aging

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Changes in gene expression patterns are a hallmark of the aging process. Important insight into the mechanisms controlling such gene expression programs has come from the study of replicative senescence of cultured cells (eg, human diploid fibroblasts), which recapitulates many features of cells from aging individuals. This Project has traditionally studied changes in RBP expression and function during replicative senescence. It has also examined the influence of RBPs in replicative senescence by interventions to elevate or reduce RBP levels, followed by the analysis of changes in senescence-associated mRNA expression patterns. We study if a given RBP binds a senescence-associated mRNA using a variety of in vitro binding assays (biotin pulldown, RNA EMSA, surface plasmon resonance/Biacore, etc) and assays to measure binding of endogenous molecules ribonucleoprotein immunoprecipitation (RIP) or crosslinking IP (CLIP). To investigate RBP function during senescence, we employ approaches such as RBP silencing, RBP overexpression, mutant RBP analysis, and RBP-associated mRNA identification (using microarrays, RNAseq, and RT-qPCR). To investigate whether RBPs affect the stability of target mRNAs during senescence, we measure the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative association of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs and use various senescence-associated markers to examine changes in the senescence phenotype. During the past funding period, we have pursued several projects to study RBP expression in aging and age-related processes. In one of the projects, we describe new findings of the RBP HuR in breast cancer (Heinonen et al., 2011) and in pancreatic cancer (Pineda et al., Cancer Biol Ther, 2012). The HuR-regulated protein sirtuin 1 (SIRT1) is implicated in survival and health span (Minor et al., Science Reports, 2011), and another HuR target, p16, is controlled via methylation of the 3-untranslated region (UTR) of the p16 mRNA (Zhang et al, Nature Commun. 2012). Another RBP implicated in cancer, hnRNP C1, was found to control the expression of PDCD4 (Park et al., Mol. Cell. Biol. 2012). During the this funding period, we reviewed the role of HuR and nucleolin in various diseases, including age-related pathologies (Srikantan and Gorospe, Frontiers in Bioscience, 2011; Abdelmohsen and Gorospe, RNA Biology, 2012), and edited a volume for the journal Aging Research Reviews, which included a review piece on RBPs affecting the production of microRNAs (Gorospe, Preface to Ageing Research Reviews, special issue) and Abdelmohsen et al., Ageing Research Reviews, 2012).