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 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 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 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 RBPs and ncRNAs 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 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. Using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis, we discovered that the RBP AUF1 (AU-binding factor 1), linked to inflammation, senescence, and aging, recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions: (1) AUF1 lowered the steady-state levels of numerous target RNAs, including long noncoding RNAs, (2) AUF1 promoted the translation of numerous mRNAs whose steady-state levels were unchanged by AUF1, and (3) AUF1 enhanced the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserved genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence (Nat Commun. 2014). Senescence-associated lncRNAs. During the past twelve months, we have also continued to investigate the influence of ncRNAs in senescence. We identified long (l)ncRNAs differentially expressed during replicative senescence by comparing lncRNAs expressed in proliferating, early-passage, young human diploid WI-38 fibroblasts population doubling (PDL) 20 with those expressed in senescent, late-passage, old fibroblasts (PDL 52) by RNA sequencing (RNASeq). Among the novel senescence-associated lncRNAs (SAL-RNAs) showing lower abundance in senescent cells, SAL-RNA1 (XLOC_023166) was found to delay senescence, because reducing SAL-RNA1 levels enhanced the appearance of phenotypic traits of senescence, including an enlarged morphology, positive b-galactosidase activity, and heightened p53 levels. These results suggested that SAL-RNAs play direct regulatory roles in this important cellular process (Abdelmohsen et al., Aging Cell 2013). In another study, we found that the SAL-RNA HOTAIR was an inducer of ubiquitin-mediated proteolysis. HOTAIR associated with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitated the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. Since HOTAIR levels were highly upregulated in senescent cells, it caused rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results, reported in Nature Communications (Yoon et al., 2013), uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination. We have also reported a number of specific microRNAs differentially expressed in senescent cells and have reviewed their interaction with lncRNAs (Yoon et al., Seminars in Cell and Developmental Biology, 2014). Senescence-associated microRNAs. During the past twelve months, we have also continued to investigate the influence of microRNAs implicated in senescence. Special attention was given to let-7, a senescence-upregulated microRNAs that can function as a tumor suppressor (Guo et al., PLoS ONE, 2013). Tissue aging. Although our understanding of the post-transcriptional factors that influence senescence is advancing quickly, we still know relatively little about the RBPs and ncRNAs that affect the aging process itself. During the current review period, we have identified differentially expressed miRNAs in skeletal muscle from young and old rhesus monkeys using RNA sequencing. In old muscle, several miRNAs were upregulated, including miR-451, miR-144, miR-18a and miR-15a, while a few miRNAs were downregulated, including miR-181a and miR-181b. We also examined the impact of caloric restriction (CR) on miRNA abundance by reverse transcription (RT) followed by real-time, quantitative (q)PCR analysis and found that CR rescued the levels of miR-181b and dampened the age-induced increase in miR-451 and miR-144 levels. Our results, reported in Aging (2014) reveal that there are changes in expression of known and novel miRNAs with skeletal muscle aging and that CR may reverse some of these changes to a younger phenotype. Most other studies on RBPs and ncRNAs influencing tissue aging during this review period (including HuD and AUF1) fit better within other projects and will be discussed therein.
| 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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