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). To investigate RBP function during senescence, we employ approaches such as RBP silencing, RBP overexpression, mutant RBP analysis, and RBP-associated mRNA identification (using microarrays, RNAseq, and RT-qPCR). To investigate whether RBPs 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 abundance. We investigate whether RBPs 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 RBPs 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 noncoding RNAs 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 (SASP). We discovered that the RBP NF90, which lowers the stability and translation of several mRNAs Tominaga-Yamanaka et al., Impact Aging 2012, associated with many mRNAs encoding SASP factors such as IL-6, IL-8, osteoprotegerin, GM-CSF, MCP-1, HCC4, Gro1, and Gro2. In young, proliferating fibroblasts, NF90 was abundant and inhibited the expression of mRNAs encoding SASP factors;by contrast, NF90 was low in senescent cells, allowing the levels of SASP factors MCP-1, GROa, IL-6, and IL-8 to rise. In collaborative studies, we have continued to investigate the roles of RBPs HuR and AUF1 in senescence, finding that the decreased nuclear export of HuR mRNA by HuR was linked to the loss of HuR in replicative senescence, that the interaction of HuR and AUF1 with the 3UTR (untranslated region) of p16 mRNA was inhibited when NSun2 methylated the p16 3UTR, leading to a rise in p16 levels Zhang et al., Nat Commun. 2012, and that the senescence-associated decline in CARM1, which methylates and hence activates HuR, was associated with the loss of HuR function in senescent cells Pang et al., BMC Mol Biol 2013. Senescence-associated ncRNAs. During the past twelve months, we have also continued to investigate the influence of ncRNAs in senescence. We have reported a number of specific microRNAs differentially expressed in senescent cells. During this evaluation period, we have also contributed several reviews to this emerging field, covering the topics of microRNAs that influence senescence and tumor suppression, the regulation of microRNA biogenesis in senescence, and the global influence of microRNAs on gene expression patterns in senescence Abdelmohsen et al., Ageing Res Reviews 2012. We have also initiated efforts to investigate the role of long ncRNAs (lncRNA) in senescence. A recent high-throughput search using RNA-Seq in collaboration with Dr. Beckers core facility, revealed numerous lncRNAs that are differentially expressed during cellular senescence. Among these senescence-associated lncRNAs, termed SAL-RNAs Abdelmohsen et al., Impact Aging 2013, we identified several transcripts that played a direct role in cellular senescence. SAL-RNAs are the subject of many follow-up studies in our laboratory that will be reported in the near future. In addition, we collaborated with the Prasanth laboratory in reporting that the lncRNA MALAT1 promoted cell proliferation and that silencing MALAT1 triggered cellular senescence Tripathi et al., PLoS Genetics, 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. We recently reported that the levels of several RBPs that regulate translation and stability (HuR, AUF1, TIA-1, and TTP) in human tissues revealed that these RBPs are abundantly expressed in most tissues in all age groups. Expression was widespread in all normal tissues, including nondividing, terminally differentiated tissues, suggesting that these RBPs play life-long physiologic roles in tissue homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000393-06
Application #
8736556
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$613,231
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Xu, Fang; Pang, Lijun; Cai, Xiaoyu et al. (2014) let-7-repressesed Shc translation delays replicative senescence. Aging Cell 13:185-92
Yoon, Je-Hyun; Abdelmohsen, Kotb; Gorospe, Myriam (2013) Posttranscriptional gene regulation by long noncoding RNA. J Mol Biol 425:3723-30
Abdelmohsen, Kotb; Panda, Amaresh; Kang, Min-Ju et al. (2013) Senescence-associated lncRNAs: senescence-associated long noncoding RNAs. Aging Cell 12:890-900
Xiao, Lan; Rao, Jaladanki N; Zou, Tongtong et al. (2013) miR-29b represses intestinal mucosal growth by inhibiting translation of cyclin-dependent kinase 2. Mol Biol Cell 24:3038-46
Pang, Lijun; Tian, Haiyan; Chang, Na et al. (2013) Loss of CARM1 is linked to reduced HuR function in replicative senescence. BMC Mol Biol 14:15
Brennan, Sarah E; Kuwano, Yuki; Alkharouf, Nadim et al. (2009) The mRNA-destabilizing protein tristetraprolin is suppressed in many cancers, altering tumorigenic phenotypes and patient prognosis. Cancer Res 69:5168-76
Kuwano, Yuki; Rabinovic, Ariel; Srikantan, Subramanya et al. (2009) Analysis of nitric oxide-stabilized mRNAs in human fibroblasts reveals HuR-dependent heme oxygenase 1 upregulation. Mol Cell Biol 29:2622-35