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 (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 (biotin pulldown, RNA EMSA, etc) 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, and RT-qPCR). 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 examined the changes in gene expression 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 CELL SURFACE MARKERS. 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. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell-surface DPP4 preferentially sensitized senescent, but not dividing fibroblasts to cytotoxicity by natural killer cells. These findings, supporting the notion that selective expression of DPP4 on the surface of senescent cells enables their preferential elimination, were reported recently (Kim et al., in press, Genes & Development, 2017). SENESCENCE-ASSOCIATED circRNAs. A new area of study was initiated in earnest during this review cycle: the analysis of circular (circ)RNAs associated with the senescent program. We began by cataloging senescence-associated circular RNAs (SAC-RNAs) and identifying Circ-PVT1 as a circRNA that suppressed senescence by sequestering the microRNA let7 (Panda et al., Nucleic Acids Res 2016), then reported methods to detect SAC-RNAs by RT-qPCR analysis (Panda et al., Methods in Molecular Biology, 2016). SENESCENCE-ASSOCIATED microRNAs. During the past twelve months, we have continued to investigate the influence of ncRNAs in senescence. During this review cycle, we have looked at microRNAs more closely. In collaboration with LEPS, we examined the role of metformin in the production of DICER1, a key protein in the biosynthesis of microRNAs, in senescent cells (Noren Hooten et al., Aging Cell, 2016). Together with the Slack team, we identified microRNAs associated with longevity using samples from the Baltimore Longitudinal Study of Aging (BLSA) (Smith-Vikos et al., Aging, 2016). The impact of miR-16 on the production of BAFF, a protein component of the senescence-associated secretory phenotype (SASP) was examined in collaboration with investigators in the SardiNIA consortium (Steri et al., The New England Journal of Medicine, 2017). Other SASP regulators were reviewed (Panda et al., Mechanisms of Ageing and Development, 2017). SENESCENCE-ASSOCIATED RBPs. Following a long-established line of research in our group, we have continued the characterization of 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. In collaboration with the Wang laboratory, we have investigated RBPs that methylate RNA via methylation at m6C and m5C (METTL3, METTL14, NSUN2) (Cai et al., Oncotarget, 2016; Li et al., J. Cell Biochem. 2017).