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RNA结合蛋白（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。 我们已经研究了给定的RBP是否使用多种体外结合测定（例如，使用生物素化的RNA片段和反义寡聚物的下拉）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。 近年来，我们分析了影响衰老和老化的非编码RNA（microRNA，mi）RNA、长链非编码RNA（lnc）RNA和环状RNA（circ）。 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、其过表达、分析或突变RBP/ncRNA以及RBP/ncRNA相关RNA鉴定（使用微阵列、RNAseq、Oxford Nanopore和RT-qPCR分析）等方法。 我们采用组织微阵列来鉴定衰老细胞并检测组织中RBP表达作为衰老和老化的函数。 为了研究RBP和ncRNA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/ncRNA丰度的函数。 我们研究是否RBP和ncRNA影响翻译的目标mRNA的mRNA的翻译，通过研究的相对关联的mRNA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。 我们还采用报告构建体，以获得额外的洞察力的RBP和ncRNA调制的过程，并使用各种衰老相关的标志物来检查衰老表型的变化。 在过去的12个月里，该项目继续研究作为生理老化一部分的人体组织中基因表达程序的变化。 我们在这个项目中的大部分努力都是为了了解不同类型的蛋白质（包括RBP）和ncRNA如何影响细胞衰老的过程，这越来越多地被认为是组织生理学和病理学中与年龄相关的潜在变化。 本项目的研究检查了调节细胞衰老的蛋白质和RNA以及它们对衰老表型的影响的后果。 在用于这些研究的细胞系统中，人二倍体成纤维细胞特别有用。 衰老相关性间皮瘤 随着越来越多的证据表明衰老组织中的衰老细胞积累与年龄相关疾病和功能下降有关，我们已经开始努力选择性地去除衰老细胞。 我们报道了RNA结合蛋白GRSF 1的丧失，如在衰老细胞中所见，通过mTOR激活信号传导以引发促炎转录程序，其包括IL 6表达水平的稳健诱导（Noh等人，Nucleic Acids Research，2019）。 我们通过对人二倍体成纤维细胞和内皮细胞中8种不同的衰老模型进行RNA测序分析，确定了在衰老中共同调控的RNA亚群（HUVEC，HAEC）通过复制耗竭、暴露于电离辐射或多柔比星以及癌基因的表达;在所有衰老模型中，50种RNA持续升高，18种RNA持续降低，包括许多蛋白质编码mRNA和一些非编码RNA（Casella et al. Nucleic Acids Research，2019）。 我们系统地鉴定了组织阵列中的p16和p21阳性细胞，所述组织阵列设计为包括来自广泛年龄范围的人的正常器官（皮肤、脑、肝、脾、肠、肺、肌肉等），并发现不同器官显示不同水平的衰老蛋白p16和p21作为年龄的函数（Idda等人，2020年老龄化）。 我们讨论了报道的lncRNA-OIS 1在控制衰老细胞表面标志物DPP 4的产生中的作用（Munk等人，非编码RNA调查，2019），并审查了非编码RNA调节衰老和衰老中的端粒稳态（Rossi和Gorospe，分子医学趋势，2020）和血液恶性肿瘤中的环状RNA（分子生物科学前沿，2020）。 实验正在进行中，以确定其他衰老相关的膜标记，以及RNA标记（编码和非编码）的衰老。 我们还开始探索RNA修饰（甲基化）在衰老和老化中的影响（Casella et al.，Advanced Science News，2019; Casella et al，WIRES RNA 2019）。 衰老相关RBP 在我们小组长期建立的研究路线之后，我们继续表征了与细胞衰老有关的几种RBP，包括增殖丧失，对应激的反应能力受损以及衰老相关分泌表型的实现。 在该报告期内，关注RBP NF 90作为与衰老分泌组相关的RNA表达程序的调节剂（Idda等人，Cell Cycle 2019）。 此外，我们回顾了通过质膜的衰老分解和衰老停滞的主题（关于“衰老和长寿中的衰老分解”的卷，丛书“健康衰老和长寿”; Kim等人，2020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 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).