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

转录后基因调控对细胞衰老的影响

• 批准号: 10688797
• 负责人: Myriam Gorospe
• 金额: $ 282.35万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688797
• 关键词: Adopted; Affect; Age; Aging; Apoptosis; Apoptosis Regulation Gene; Atherosclerosis; Attention; Binding; Binding Proteins; Biological Assay; Biopsy; Blood Vessels; Cardiovascular system; Cell Aging; Cell physiology; Cells; Cultured Cells; Cyclin-Dependent Kinase Inhibitor; Cyclins; Diploidy; Disease; Fibroblasts; Gene Expression; Gene Expression Profile; Genetic Transcription; Growth Factor; Human; Immunoprecipitation; In Vitro; Individual; Inflammation; Intervention; Link; Longevity; Malignant Neoplasms; Measures; Messenger RNA; Metalloproteases; MicroRNAs; Muscular Atrophy; Nucleic Acids; Oncoproteins; Participant; Pathologic; Pathology; Pattern; Phenotype; Physiological; Physiology; Polyribosomes; Post-Transcriptional Regulation; Process; Proteins; Proteome; RNA; RNA Interference; RNA-Binding Proteins; Reporter; Reporting; Research; Ribonucleoproteins; Role; Science; Skin; System; Tissue Microarray; Tissues; Translating; Translations; Tumor Suppressor Proteins; Untranslated RNA; Work; age related; base; cell injury; circular RNA; cohort; crosslink; cytokine; experimental study; extracellular vesicles; human tissue; insight; interest; lamin B receptor; mRNA Expression; mouse model; muscle form; muscle regeneration; mutant; nanopore; overexpression; programs; protein biomarkers; protein expression; protein function; response; senescence; transcriptome; transcriptome sequencing

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.

基因表达模式的变化是衰老过程的标志。对控制此类基因表达程序的机制的重要洞察来自研究培养细胞的复制衰老（例如，人二倍成纤维细胞），这些细胞概括了衰老个体的细胞的许多特征。该项目传统上研究了在复制衰老过程中RNA结合蛋白（RBP）表达和功能的变化。它还通过干预措施检查了RBP在复制衰老中的影响，以升高或降低RBP水平，然后分析与衰老相关的mRNA表达模式的变化。我们已经研究了给定的RBP是否使用各种体外结合测定（例如，使用生物素化的RNA片段和反义寡聚物）结合衰老相关的mRNA，并测量内源性分子的结合内源分子的核糖核酸蛋白核蛋白免疫蛋白免疫原蛋白（RIP）或Crosslip linking（Clip）。近年来，我们包括对影响衰老和衰老的未编码RNA MicroRNA（MI）RNA，长非编码（LNC）RNA和圆（CIRC）RNA的分析。 为了研究衰老过程中的RBP和NCRNA功能，我们采用了诸如RBP或NCRNA的沉默，相同的过表达，分析或突变体RBPS/NCRNA以及RBP/NCRNA相关的RNA鉴定（使用微阵列，使用微阵列，RNASEQ，RNASEQ，RNASEQ，OXFORD NANANOPORE和RT-QPCR ANALYSES）。我们采用了组织微阵列来鉴定衰老细胞并检测组织中的RBP表达是衰老和衰老的函数。为了研究RBP和NCRNA是否会影响衰老过程中目标mRNA的稳定性，我们测量了感兴趣的mRNA的稳态水平和半衰期作为RBP/NCRNA丰度的函数。我们研究了RBP和NCRNA是否通过研究mRNA与翻译多粒子体的相对关联以及量化编码蛋白的新生翻译速率来影响靶mRNA的翻译。我们还采用记者构造来获得对RBPS和NCRNA调节过程的进一步见解，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个月中，该项目继续研究人体组织中发生的基因表达程序的变化，这是生理衰老的一部分。我们在该项目中的大部分努力都旨在了解不同类型（包括RBP）和NCRNA的蛋白质如何影响细胞衰老的过程，细胞衰老的过程越来越被认为是组织生理和病理学与年龄相关的潜在变化。该项目的研究检查了调节细胞衰老的蛋白质和RNA及其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。 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。 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). 我们还分类了从Gestalt参与者那里取的皮肤活检的成纤维细胞的蛋白质组织（Tsitsipatis等人，Aging Cell，2021）。 正在进行实验，以确定DPP4在动脉粥样硬化的小鼠模型中，衰老组的皮肤成纤维细胞的转录组以及PatchD4，AnkRD1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存在的蛋白质标记，以及衰老在肌肉再生中的作用。