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 (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, AGO2 let-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). Along these lines, the Wilce laboratory (Monash University, Melbourne , Australia), collaborated with our group to characterize the collaborative interplay of let-7 binding to the MYC mRNA, which encodes another senescent modulatory protein (MYC) (Gunzburg et al., Cell Cycle, 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 (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; Abdelmohsen et al., Aging) A new class of RBPs affecting senescence-associated gene expression contains RNA methyltransferases. In particular, the methyltransferase NSUN2 is repressed as cells become senescent. As the Wang laboratory (Beijing University) found in collaboration with our lab, the reduced abundance of NSUN2 in senescent fibroblasts and HUVEC cells leds to a reduction in the levels and function of several microRNAs, including miR-125b, elevated the production of the cyclin-dependent kinase (CDK) inhibitor p27, and lowered production of CDK1 (Xing et al., Molecular and Cellular Biology, 2015; Tang et al., Aging 2016; Cai et al., Oncotarget 2016). 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, the Evans laboratory (LEPS, NIA) reported, in collaboration with our group, that expression of DICER1, a key enzyme in the biosynthesis of microRNAs, is repressed by the RNA-binding protein AUF1. With the decline of AUF1 in senescent cells and in response to metformin treatment, DICER1 levels rise and the production of several senescence- and aging-associated microRNAs rises (Noren Hooten et al., Aging, 2016).
| 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 |
| Kim, Jiyoung; Noh, Ji Heon; Lee, Seung-Kyu et al. (2017) LncRNA OIP5-AS1/cyrano suppresses GAK expression to control mitosis. Oncotarget 8:49409-49420 |
| Abdelmohsen, Kotb; Panda, Amaresh C; Munk, Rachel et al. (2017) Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1. RNA Biol 14:361-369 |
| Li, Qiu; Li, Xiu; Tang, Hao et al. (2017) NSUN2-Mediated m5C Methylation and METTL3/METTL14-Mediated m6A Methylation Cooperatively Enhance p21 Translation. J Cell Biochem 118:2587-2598 |
| Kim, Kyoung Mi; Noh, Ji Heon; Bodogai, Monica et al. (2017) Identification of senescent cell surface targetable protein DPP4. Genes Dev 31:1529-1534 |
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