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(如人的二倍体成纤维细胞)的复制衰老的研究，人们对这种基因表达程序的调控机制有了重要的认识，它概括了衰老个体细胞的许多特征。该项目传统上研究复制衰老过程中RNA结合蛋白(RBP)表达和功能的变化。它还通过提高或降低RBP水平的干预，研究了限制性商业惯例在复制衰老中的影响，随后分析了与衰老相关的mRNA表达模式的变化。我们已经使用各种体外结合分析(例如，使用生物素化RNA片段和反义寡聚物的下拉)以及测量内源分子核糖核蛋白免疫沉淀(RIP)或交联型IP(CLIP)的结合试验，研究了给定的RBP是否与衰老相关的mRNA结合。近年来，我们包括了对影响衰老的非编码RNA、微RNAs(MI)RNAs、长非编码(LNC)RNAs和环状(CIRC)RNAs的分析。为了研究RBP和ncRNAs在衰老过程中的功能，我们采用了一些方法，如沉默RBP或ncRNA，过表达，分析或突变的rBPs/ncRNAs，以及RBP/ncRNA相关的RNA鉴定(使用微阵列、RNAseq、牛津纳米孔和RT-qPCR分析)。我们已经采用组织微阵列来识别衰老细胞，并检测RBP在组织中的表达作为衰老和衰老的函数。为了研究RBPs和ncRNAs是否影响靶mRNAs在衰老过程中的稳定性，我们测量了目标mRNAs的稳态水平和半衰期作为RBP/ncRNA丰度的函数。我们通过研究mRNA与翻译多聚体的相对关联以及通过量化编码蛋白的新生翻译率来研究RBPs和ncRNAs是否影响目标mRNAs的翻译。我们还使用报告结构来进一步了解RBPs和ncRNAs调控的过程，并使用各种与衰老相关的标记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衰老的一部分，在人体组织中发生的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(包括限制性商业惯例)和ncRNAs如何影响细胞衰老过程，这一过程越来越被认为是组织生理和病理中与年龄相关的潜在变化。该项目的研究考察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。在用于这些研究的细胞系统中，人类二倍体成纤维细胞提供了特别多的信息。 衰老相关转录组 随着越来越多的证据表明衰老组织中衰老细胞的积累与年龄相关的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。我们报告了在衰老细胞中看到的RNA结合蛋白GRSF1的丢失，通过mTOR激活信号以引发促炎转录程序，其中包括强劲地诱导IL6的表达水平(Noh等人，核酸研究，2019年)。我们通过对八种不同的衰老模型进行RNA测序分析，确定了共同调节衰老的RNA亚群；在所有衰老模型中，50个RNA持续升高，18个RNA持续减少，包括许多蛋白质编码RNA和一些非编码RNA(Casella等人)。《核酸研究》，2019年)。我们在包括正常器官(皮肤、脑、肝、脾、肠、肺、肌肉等)的组织阵列中系统地鉴定了来自不同年龄段的人的p16和p21阳性细胞，并发现不同的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，Aging 2020)。 我们讨论了已报道的lncRNA-OIS1在控制衰老细胞表面标记DPP4的产生中的作用(Munk等人，非编码RNA调查，2019年)，并综述了在衰老和衰老中调节端粒稳态的非编码RNA(Rossi和Gorospe，2020年)和血液恶性肿瘤中的环状RNA(分子生物科学前沿，2020)。 目前正在进行实验，以确定其他与衰老相关的膜标记，以及衰老的RNA标记(编码和非编码)。我们还开始探索RNA修饰(甲基化)对衰老和衰老的影响(Casella等人，2019年《高级科学新闻》；Casella等人，Wire RNA 2019)。 衰老相关的限制性商业惯例 根据我们小组长期确立的研究路线，我们继续描述了几种与细胞衰老有关的限制性商业惯例的特征，包括增殖丧失，对压力的反应能力受损，以及实现与衰老相关的分泌表型。在本报告所述期间，重点研究了RBP NF90作为与衰老分泌体相关的RNA表达程序的调节器(Idda等人，细胞周期2019)。此外，我们通过质膜回顾了感受器分解和感受器稳定的主题(关于“衰老和长寿中的感受剂”卷，丛书“健康的衰老和长寿”；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).