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。 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。 在本综述期间进行的工作的实例中，我们已经鉴定了miR-340- 3 p通过减少核纤层蛋白B受体（LBR）的丰度触发衰老（赫尔曼等人，Nucleic Acids Research，2021），并报道了SRC活化是使受损细胞的反应从凋亡向衰老倾斜所必需的（Anerillas et al.，Science Advances 2022）。 我们还对取自GESTALT参与者的皮肤活组织检查的成纤维细胞的蛋白质组进行了编目（Tsitsipatis等人，Aging Cell，2021）。 实验正在进行中，以确定DPP 4在动脉粥样硬化小鼠模型中衰老血管细胞中的作用，来自GESTALT队列的皮肤成纤维细胞的转录组，以及PATCHD 4，ANKRD 1和BAFF在衰老中的作用。 我们还开始研究衰老细胞分泌的细胞外囊泡上的蛋白质标记物，以及衰老在肌肉再生中的作用。

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. 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. In examples of the work performed during this review period, we have identified miR-340-3p as triggering senescence by reducing the abundance of lamin B receptor (LBR) (Herman et al., Nucleic Acids Research, 2021), and reported that SRC activation is required to tilt the response of damaged cells from apoptosis to senescence (Anerillas et al., Science Advances 2022). We also catalogued the proteomes of fibroblasts from skin biopsies taken from GESTALT participants (Tsitsipatis et al., Aging Cell, 2021). Experiments are underway to identify the role of DPP4 in senescent vascular cells in a mouse model of atherosclerosis, the transcriptomes of skin fibroblasts from the GESTALT cohort, and the roles of PATCHD4, ANKRD1, and BAFF in senescence. We have also started to look at protein markers present on extracellular vesicles secreted by senescent cells, as well as the role of senescence in muscle regeneration.