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

基因表达模式的变化是衰老过程的标志。对控制这种基因表达程序的机制的重要见解来自对培养细胞（例如，人二倍体成纤维细胞）的复制衰老的研究，其概括了来自衰老个体的细胞的许多特征。该项目传统上研究了复制衰老过程中RBP表达和功能的变化。它还研究了RBP在复制性衰老中的影响，通过干预来提高或降低RBP水平，然后分析衰老相关mRNA表达模式的变化。我们使用各种体外结合测定（生物素下拉、RNA EMSA、表面等离子体共振/Biacore等）和测定内源性分子核糖核蛋白免疫沉淀（RIP）或交联IP（CLIP）的结合的测定来研究给定的RBP是否结合衰老相关mRNA。为了研究RBP在衰老过程中的功能，我们采用了RBP沉默、RBP过表达、突变体RBP分析和RBP相关mRNA鉴定（使用微阵列、RNAseq和RT-qPCR）等方法。为了研究RBP是否影响衰老过程中靶mRNA的稳定性，我们测量了目标mRNA的稳态水平和半衰期作为RBP丰度的函数。我们调查是否RBP影响翻译的目标mRNA的mRNA通过研究的相对关联与翻译的多核糖体，并通过量化的新生翻译率的编码蛋白质。我们还采用报告结构，以获得更多的洞察力的过程中调节的RBP和使用各种衰老相关的标记物来检查衰老表型的变化。 在过去的资助期间，我们进行了几个项目，研究衰老和年龄相关过程中的RBP表达。在其中一个项目中，我们描述了RBP HuR在乳腺癌中的新发现（Heinonen等人，2011）和胰腺癌（Pineda等人，Cancer Biol Ther，2012）。 HuR调节的蛋白sirtuin 1（SIRT 1）与存活和健康寿命有关（Minor et al.，Science Reports，2011），并且另一种HuR靶标p16通过p16 mRNA的3-非翻译区（UTR）的甲基化来控制（Zhang等人，Nature Commun. 2012年）。 另一种与癌症有关的RBP hnRNP C1被发现控制PDCD 4的表达（Park等人，摩尔Cell. 2012）。 在此资助期间，我们回顾了HuR和核仁素在各种疾病中的作用，包括与年龄相关的病理学（Srikantan和Gorospe，Frontiers in Bioscience，2011; Abdelmohsen和Gorospe，RNA Biology，2012），并为期刊Aging Research Reviews编辑了一卷，其中包括关于影响microRNA产生的RBP的评论文章（Gorospe，《老龄化研究评论》序言，特刊）和Abdelmohsen等人，老龄化研究评论，2012年）。

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).