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

National Institute On Aging

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Abstract

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 (e.g., 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), ncRNAs, and signaling pathways 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 proposed that alternative splicing influences senescence and aging (Ferrucci et al., Nature Aging 2022), have found that the kinase SRC, and the signaling paradigms converging on BDNF-TrkB and YAP-TEAD promote survival during cell senescence (Anerillas et al., Science Advances 2022, Anerillas et al., Nature Communications 2022, and Nature Aging 2023). We also catalogued the proteomes and transcriptomes of fibroblasts from skin biopsies taken from GESTALT participants (Tsitsipatis et al., Aging Cell, 2022 and Aging Cell 2023). We identified the role of DPP4 in senescent vascular cells in a mouse model of atherosclerosis (Herman et al., Journal of Clinical Investigation 2023) and the role of BAFF in senescence (Rossi et al., 2023). We have started to actively look at protein markers present on extracellular vesicles and particles secreted by old organs (Abdelmohsen et al., Journal of Extracellular Biology 2023) and senescent cells, as well as the role of senescence in muscle regeneration.