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). Following up on this investigation, we recently discovered that the AUF1 isoform p37 had very strong affinity for the microRNA let-7b in vitro. A variety of in vitro and in vivo analyses showed that AUF1 promoted the loading of let-7b onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2let-7 triggered target mRNA decay. This novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing was recently reported in Genes and Development (Yoon et al., 2015). Senescence-associated lncRNAs. During the past twelve months, we have also continued to investigate the influence of ncRNAs in senescence. We had previously 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). Some of these SAL-RNAs appeared to play direct regulatory roles in this important cellular process (Abdelmohsen et al., Aging Cell 2013). Given the accumulation of reports on lncRNAs influencing senescence and aging, we were invited to prepare a number of reviews on this topic. Accordingly, the past twelve months have been filled with requests to review this area of aging biology (Yoon et al., Seminars in Cell and Developmental Biology, 2014; Grammatikakis et al., Aging 2015; Greco et al., Journal of Molecular and Cellular Cardiology, 2015; Kim et al., Biochimica et Biophysica Acta, 2015; Abdelmohsen and Gorospe, WIRES RNA, 2015; One of the lncRNAs that does not show differential abundance with senescence, but does show differential function with senescence is 7SL. During this review period, we obtained evidence that 7SL formed a partial hybrid with the 3'-untranslated region (UTR) of TP53 mRNA, which encodes the tumor suppressor and senescence regulator p53. The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes. Silencing 7SL led to increased binding of HuR to TP53 mRNA, an interaction that led to the promotion of p53 translation and increased p53 abundance. These results, reported by Abdelmohsen et al., (Nucleic Acids Res 2014) suggest that the competition between 7SL and HuR for binding to TP53 3'UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53. Our findings further suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels. 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 collaborated with Dr. Kwons laboratory to identify microRNA miR-431 as a novel miRNA showing markedly reduced abundance in aged myoblasts. Our studies showed that elevating miR-431 improved the myogenic capacity of old myoblasts, while inhibiting endogenous miR-431 lowered myogenesis. miR-431 interacted with the 3'UTR of Smad4 mRNA, which encodes one of the downstream effectors of TGF-β signaling. In agreement with the low levels of miR-431 in old myoblasts, SMAD4 levels increased in this myoblast population. In an in vivo model of muscle regeneration following cardiotoxin injury, ectopic miR-431 injection greatly improved muscle regeneration and reduced SMAD4 levels. Moreover, inhibition of miR-431 also repressed the myogenic capacity of human skeletal myoblasts. This work reveals that the age-associated miR-431 plays a key role in maintaining the myogenic ability of skeletal muscle with age (Lee et al., Genes and Development, 2015).
| 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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