Influence of Post-transcriptional Gene Regulation on Cell Senescence and Aging

基因表达模式的变化是衰老过程的一个标志。通过对培养细胞（如人类二倍体成纤维细胞）的复制性衰老的研究，对控制这些基因表达程序的机制有了重要的认识，这些细胞概括了衰老个体细胞的许多特征。本项目主要研究复制衰老过程中rna结合蛋白（RBP）表达和功能的变化。通过干预提高或降低RBP水平，研究了RBP对复制性衰老的影响，随后分析了衰老相关mRNA表达模式的变化。我们已经研究了一个给定的RBP是否与衰老相关的mRNA结合，使用各种体外结合测定（例如，使用生物素化RNA片段和反义寡聚物）和测定内源性分子的结合，核糖核蛋白免疫沉淀（RIP）或交联IP （CLIP）。近年来，我们对影响衰老和衰老的非编码rna （microRNAs (mi) rna）、长链非编码rna （lnc）和环状rna （circ）进行了分析。为了研究RBP和ncRNA在衰老过程中的功能，我们采用了诸如RBP或ncRNA沉默，相同的过表达，分析或突变RBP/ncRNA以及RBP/ncRNA相关RNA鉴定（使用微阵列，RNAseq，牛津纳米孔和RT-qPCR分析）等方法。我们采用组织微阵列技术鉴定衰老细胞，检测组织中RBP的表达作为衰老和衰老的功能。为了研究RBP和ncRNA是否会影响衰老过程中目标mrna的稳定性，我们测量了RBP/ncRNA丰度对目标mrna的稳态水平和半衰期的影响。我们通过研究mRNA与翻译多体的相对关联以及量化编码蛋白的初始翻译速率来研究rbp和ncRNAs是否影响目标mRNA的翻译。我们还采用报告者结构来进一步了解rbp和ncrna调节的过程，并使用各种衰老相关标记来检查衰老表型的变化。

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 RNA-binding protein (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 have studied if a given RBP binds a senescence-associated mRNA using a variety of in vitro binding assays (e.g., pulldown using biotinylated RNA segments and antisense oligomers) and assays to measure binding of endogenous molecules ribonucleoprotein immunoprecipitation (RIP) or crosslinking IP (CLIP). In recent years, we have included the analysis of noncoding RNAs microRNAs (mi)RNAs, long noncoding (lnc)RNAs, and circular (circ)RNAs that influence senescence and aging. To investigate RBP and ncRNAs function during senescence, we employ approaches such as silencing of the RBP or ncRNA, overexpression of the same, analysis or mutant RBPs/ncRNAs, and RBP/ncRNA-associated RNA identification (using microarrays, RNAseq, Oxford Nanopore, and RT-qPCR analyses). We have adopted tissue microarrays to identify senescent cells and detect RBP expression in tissues as a function of senescence and aging. To investigate whether RBPs and ncRNAs 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/ncRNA abundance. We investigate whether RBPs and ncRNAs 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 the RBPs and ncRNAs and use various senescence-associated markers to examine changes in the senescence phenotype. Over the past 12 months, this Project has continued to examine changes in gene expression programs that occur in human tissues as part of physiologic aging. Much of our effort in this Project has been directed at understanding how proteins of different types (including RBPs) and ncRNAs affect the process of cellular senescence, which is increasingly recognized as underlying age-related changes in tissue physiology and pathology. The studies in this Project examine the proteins and RNAs that modulate cellular senescence and the consequences of their influence on the senescent phenotype. Among the cell systems used for these studies, human diploid fibroblasts have been particularly informative. SENESCENCE-ASSOCIATED TRANSCRIPTOME With increasing evidence that senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, we have initiated efforts to remove senescent cells selectively. We reported that loss of the RNA-binding protein GRSF1, as seen in senescent cells, activates signaling through mTOR to elicit a proinflammatory transcriptional program that includes a robust induction of IL6 expression levels (Noh et al., Nucleic Acids Research, 2019). We identified subsets of RNAs jointly regulated in senescence by performing RNA-sequencing analysis across eight diverse models of senescence triggered in human diploid fibroblasts and endothelial cells (HUVEC, HAEC) by replicative exhaustion, exposure to ionizing radiation or doxorubicin, and expression of an oncogene; 50 RNAs were consistently elevated and 18 RNAs consistently reduced across all senescence models, including many protein-coding mRNAs and some non-coding RNAs (Casella et al. Nucleic Acids Research, 2019). We identified systematically p16 and p21positive cells in tissue arrays designed to include normal organs (skin, brain, liver, spleen, intestine, lung, muscle, etc) from persons across a broad spectrum of ages and found that different organs display different levels of the senescent proteins p16 and p21 as a function of age (Idda et al., Aging 2020). We discussed the reported role for lncRNA-OIS1 in controlling the production of senescent cell surface marker DPP4 (Munk et al., Noncoding RNA Investigation, 2019), and reviewed noncoding RNAs modulating telomere homeostasis in senescence and aging (Rossi and Gorospe, Trends in Molecular Medicine, 2020) and circular RNAs in blood malignancies (Frontiers in Molecular Biosciences, 2020). Experiments are underway to identify other senescence-associated membrane markers, as well as RNA markers (coding and noncoding) of senescence. We have also begun to explore the impact of RNA modifications (methylation) in senescence and aging (Casella et al., Advanced Science News, 2019; Casella et al, WIRES RNA 2019). SENESCENCE-ASSOCIATED RBPs Following a long-established line of research in our group, we have continued the characterization of several RBPs implicated in aspects of cellular senescence, including the loss of proliferation, the impaired ability to respond to stress, and the implementation of a senescence-associated secretory phenotype. Within this reporting period focused on the RBP NF90 as a regulator of RNA expression programs related to the senescence secretome (Idda et al., Cell Cycle 2019). Additionally, we reviewed the topics of senolysis and senostasis through the plasma membrane (volume on "Senolytics in Ageing and Longevity", book series "HEALTHY AGEING AND LONGEVITY"; Kim et al., 2020).