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 continue 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. Mass spectrometry analysis revealed that secretory carrier membrane protein 4 (SCAMP4) levels were strikingly elevated on the surface of senescent cells compared with proliferating cells. Interestingly, silencing SCAMP4 in senescent fibroblasts reduced the secretion of SASP factors, including interleukin 6 (IL6), IL8, growth differentiation factor 15 (GDF-15), C-X-C motif chemokine ligand 1 (CXCL1), and IL7, while, conversely, SCAMP4 overexpression in proliferating fibroblasts increased SASP factor secretion. Our results indicate that SCAMP4 accumulates on the surface of senescent cells, promotes SASP factor secretion, and critically enhances the SASP phenotype (Kim et al., Genes & Development, 2018). We have also identified subsets of RNAs jointly regulated across different models of senescence (Casella et al. Nucleic Acids Research, 2019) and a role for lncRNA-OIS1 in controlling the production of senescent cell surface marker DPP4 (Munk et al., Noncoding RNA Investigation, 2019). We have also initiated a new area of study focused on the impact of RNA modifications (methylation) in senescence and aging (Casella et al., Advanced Science News, 2019; Casella et al, WIRES RNA 2019). Experiments are underway to identify other senescence-associated membrane markers, as well as RNA markers (coding and noncoding) of senescence. 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. Two studies within this reporting period focused on the RBP NF90 as a regulator of RNA expression programs related to the senescence secretome (Noh et al., Aging 2018; Idda et al., Nucleic Acids Research 2018; Idda et al., Cell Cycle 2019).
| Omer, Amr; Patel, Devang; Lian, Xian Jin et al. (2018) Stress granules counteract senescence by sequestration of PAI-1. EMBO Rep 19: |
| Kim, Kyoung Mi; Noh, Ji Heon; Bodogai, Monica et al. (2018) SCAMP4 enhances the senescent cell secretome. Genes Dev 32:909-914 |
| Tang, Hao; Wang, Hu; Cheng, Xiaolei et al. (2018) HuR regulates telomerase activity through TERC methylation. Nat Commun 9:2213 |
| Tang, Hao; Wang, Hu; Cheng, Xiaolei et al. (2018) Author Correction: HuR regulates telomerase activity through TERC methylation. Nat Commun 9:2721 |
| Noh, Ji Heon; Kim, Kyoung Mi; McClusky, Waverly G et al. (2018) Cytoplasmic functions of long noncoding RNAs. Wiley Interdiscip Rev RNA 9:e1471 |
| Basu, Sandip K; Gonit, Mesfin; Salotti, Jacqueline et al. (2018) A RAS-CaMKK?-AMPK?2 pathway promotes senescence by licensing post-translational activation of C/EBP? through a novel 3'UTR mechanism. Oncogene 37:3528-3548 |
| Lee, Hyung Chul; Jung, Seung Hee; Hwang, Hyun Jung et al. (2017) WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms. Nucleic Acids Res : |
| Munk, Rachel; Panda, Amaresh C; Grammatikakis, Ioannis et al. (2017) Senescence-Associated MicroRNAs. Int Rev Cell Mol Biol 334:177-205 |
| Panda, Amaresh C; Grammatikakis, Ioannis; Kim, Kyoung Mi et al. (2017) Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1. Nucleic Acids Res 45:4021-4035 |
| Steri, Maristella; Orrù, Valeria; Idda, M Laura et al. (2017) Overexpression of the Cytokine BAFF and Autoimmunity Risk. N Engl J Med 376:1615-1626 |
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