| RNA Regulation Section |
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Myriam Gorospe, Ph.D., Chief |
| Overview |
| Research Summary:Aging is characterized by a general decline in the ability of individuals to respond adequately to different damaging and proliferative agents. Changes in the expression of many stress-response and mitogenic proteins are believed to play an important role in determining cell fate. Although this regulation has a significant transcriptional component, it is now well established that post-transcriptional mechanisms also critically influence gene expression during the stress response and cell division. This post-transcriptional control is primarily elicited through changes in the stability of the mRNA and its translation rate, but can also be modulated by events such as pre-mRNA splicing, nuclear export of the mRNA, and localization of cytoplasmic mRNA. Our long-term efforts are three-fold: (1) to investigate the turnover and translation of specific mRNAs, (2) to investigate the mRNA-binding factors [RNA-binding proteins (RBPs) and microRNAs (miRNAs)] involved in mRNA turnover and translation, and (3) to investigate the influence of mRNA turnover and translation on biological processes. Below are examples of ongoing and recently completed studies in these areas: |
| MKP-1 mRNA stabilization and translational control by RBPs: The mitogen-activated protein (MAP) kinase phosphatase (MKP)-1 dephosphorylates and thereby inactivates MAP kinases, particularly JNK (c-Jun N-terminal kinase) and p38. We have examined the post-transcriptional events responsible for the induction of MKP-1 by oxidants in HeLa cells. Evidence that H2O2 treatment potently stabilized the MKP-1 mRNA and increased its association with heavy polysomes led us to hypothesize that the association of MKP-1 mRNA with RBPs might influence the half-life and translation of the MKP-1 mRNA. Four RBPs which influence mRNA turnover and/or translation (HuR, NF90, TIAR, and TIA-1) bound to biotinylated transcripts spanning the MKP-1 AU-rich 3’-untranslated region (UTR). By ribonucleoprotein immunoprecipitation (RNP IP) analysis, H2O2 treatment specifically increased the association of MKP-1 mRNA with HuR and NF90. Individually silencing HuR or NF90 diminished the H2O2-stimulated MKP-1 mRNA stability, indicating that both HuR and NF90 increased MKP-1 mRNA half-life. In addition, HuR silencing also strongly decreased MKP-1 translation. Importantly, lowering MKP-1 expression by HuR silencing (wherein MKP-1 mRNA stability and translation were reduced) led to a marked elevation in phosphorylated JNK and p38 after H2O2 treatment. By contrast, NF90 silencing appeared to enhance MKP-1 translation, suggesting that NF90 inhibited MKP-1 translation. Taken together, MKP-1 upregulation by oxidative stress is potently influenced by increased mRNA stability and translation, mediated at least in part by RBPs HuR and NF90. |
| p16INK4a translation is suppressed by miR-24: Expression of the tumor suppressor p16INK4a (p16) increases with aging and replicative senescence. However, in senescent WI-38 human diploid fibroblasts (HDFs) the marked elevation in p16 protein levels was accompanied by a much more modest changes in p16 mRNA levels. Global analysis of microRNAs expressed in HDFs revealed that the abundance of miR-24 increased dramatically with replicative senescence. Computational analysis predicted that miR-24 interacted with the p16 mRNA both in its coding region (CR) and its 3’UTR. In HDFs, ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels, while introduction of an ‘antagomir’ RNA, an antisense (AS) transcript that lowered miR-24 levels, markedly enhanced p16 protein levels, supporting the view that miR-24 suppressed p16 translation. Further experiments in HeLa cells indicated that miR-24 likely suppressed both the elongation and initiation phases of translation, based on the relative sizes of p16 mRNA-associated polyribosomes. These effects relied on both the p16 CR and 3’UTR sites of miR-24 interaction, as observed using EGFP-p16 reporters bearing miR-24 target recognition sites. Current efforts are focused on the influence of miR-24 on other key target mRNAs. |
| Regulation of HuR expression by miR-519: In addition to the control of HuR function by modifying its subcellular localization, evidence is increasing that total HuR levels are also subject to regulation. We recently identified a microRNA (miR-519) that specifically suppressed HuR expression. HuR mRNA was predicted to have two sites of miR-519 association (as determined using the software RNA22), one in the HuR CR, one in the HuR 3’UTR. In HeLa cells, miR-519 overexpression by transfection of a precursor (Pre)miR-519 reduced HuR levels, while miR-519 reduction by using antisense (AS)miR-519 elevated HuR levels. These changes in HuR abundance were not due to changes in HuR mRNA, but to altered HuR biosynthesis, as determined by measuring de novo HuR translation. In turn, the changes in HuR expression correlated with alterations in HuR RNPs containing target transcripts (e.g., cyclin B1, prothymosin α, nucleolin, and VHL mRNAs) and influenced the levels of the encoded proteins. In keeping with HuR’s proliferative influence, reductions in miR-519 increased cell proliferation and 3H-thymidine incorporation, while elevating miR-519 reduced these parameters. Efforts are underway to study the influence of miR-519 on tumorigenesis and to elucidate the extent to which miR-519 actions depend upon its effects on HuR. |
| En masse analysis of AUF1: identification of signature motif on target mRNAs: In most studies, AUF1 has been found to promote target mRNA decay, but it also stabilized and promoted the translation of specific target transcripts. To study AUF1 function, we first sought to identify AUF1 target mRNAs systematically. AUF1 was immunoprecipitated under conditions that preserved AUF1-RNA interactions, whereupon AUF1-associated target mRNAs were identified using cDNA arrays. In collaboration with Dr. Ming Zhan (Bioinformatics Unit, RRB, NIA), we studied the primary sequences and secondary structures shared among the 3’UTRs of the most highly enriched mRNAs in AUF1 IPs. Among the top candidate motifs, the motif showing the highest abundance in the experimental 3’UTR dataset compared with the entire 3’UTR database was selected. The motif was 29-39 nt long and was rich in A/U residues (~79% AU), in keeping with the sequences of many reported AUF1 target mRNAs. We tested the predictive value of the novel AUF1 motif by studying if AUF1 associated with mRNAs in which at least one AUF1 motif copy was identified computationally. Both RNP IP and biotin pulldown analyses confirmed that these putative target mRNAs did associate with AUF1; moreover, they appeared to interact with AUF1 as pre-mRNAs, as determined using nuclear lysates for RNP IP analysis and RT-qPCR employing primer pairs that spanned intron-exon junctions. Unexpectedly, silencing of AUF1 did not broadly elevate the levels of mRNAs bearing the AUF1 motif. Likewise, AUF1 overexpression did not suppress the levels of these mRNAs. Ongoing studies are testing our working hypothesis that AUF1 has little effect on target mRNAs in unstimulated cells, and instead elicits its postranscriptional influence following exposure to damaging, immune, or proliferative stimuli. |
| Ubiquitin-mediated degradation of HuR following heat shock: In response to mild heat shock (HS, 43 ºC), the levels of HuR markedly declined in a variety of cell types. In cells that were allowed to recover at 37 ºC, HuR levels returned to the original levels by ~12 h. The impact of the lowered HuR levels upon the collection of expressed transcripts was similar to that seen after siRNA-mediated silencing of HuR, as determined using microarrays. This mode of HuR regulation was unexpected, since stress agents are broadly believed to keep HuR levels unchanged and instead alter HuR cytoplasmic abundance by nucleocytoplasmic shuttling through the HuR nucleocytoplasmic shuttling (HNS) domain. Therefore, we investigated how HS reduced HuR levels. HS treatment did not lower the amount of HuR mRNA nor did it decrease the rate of de novo HuR translation. Instead, HS appeared to reduce HuR by promoting its degradation via the ubiquitin/proteasome pathway. Supporting the involvement of the ubiquitin–proteasome system in this process were results showing that (1) HuR was ubiquitinated in vitro and in intact cells, (2) proteasome inhibition increased HuR abundance after HS, and (3) the HuR kinase checkpoint kinase 2 protected against the loss of HuR by HS. Within a central, HS-labile B110-amino-acid region, K182 was found to be essential for HuR ubiquitination and proteolysis as mutant HuR(K182R) was left virtually unubiquitinated and was refractory to HS-triggered degradation. Our findings show that HS transiently lowers HuR by proteolysis linked to K182 ubiquitination and that HuR reduction enhances cell survival following HS. |
Nuclear Retention of HuR Through Phosphorylation by Cdk1: Although HuR is predominantly nuclear, it translocates to the cytoplasm in response to stress and proliferative signals. The mechanisms that regulate the subcellular location of HuR are of great interest, since HuR’s influence upon target mRNAs is linked to its presence in the cytoplasm. We recently found that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences the subcellular distribution of HuR. Endogenous HuR was specifically phosphorylated in synchronous G2-phase cultures, HuR cytoplasmic levels increased by interventions that inhibited Cdk1, and they declined in response to interventions that activated Cdk1. In keeping with the prominently cytoplasmic location of the non-phosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was found to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was further linked to its lower association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, retaining it in the nucleus in association with 14-3-3 and thereby influencing its post-transcriptional functions. |
Post-transcriptional regulation of Nitric Oxide-induced mRNAs in fibroblasts: Nitric oxide (NO) is a potent biological molecule that regulates important physiological functions such as inflammation, vascular tone, and neurotransmission. NO and its by-products cause oxidative stress and trigger signal transduction pathways leading to changes in gene expression through altered transcription, translation, and mRNA stability. Lung fibroblasts are readily exposed to NO through external sources such as smoke and diesel exhaust. We used primary human lung fibroblasts (IMR-90) and mouse NIH3T3 cells to investigate the mechanisms of post-transcriptional NO-regulated mRNA expression. Among the mRNAs that were stabilized by NO treatment, seven transcripts were found to be shared by IMR-90 and NIH3T3 cells (CHIC2, GADD45B, HO-1, PTGS2, RGS2, TIEG, ID3) and were chosen for further analysis. All seven mRNAs showed at least one hit of a signature motif for HuR, the stabilizing RBP and RNP IP analysis confirmed that all seven mRNAs associated with HuR. In keeping with a functional role of HuR in the response to NO, a measurable fraction of HuR increased in the cytoplasm following NO treatment. However, among the seven transcripts, only HO-1 mRNA showed a robust increase in its association with HuR following NO treatment. In turn, HO-1 mRNA and protein levels were significantly reduced when HuR expression was silenced in IMR-90 cells, and they were elevated when HuR was overexpressed. In sum, our results indicate that NO stabilizes mRNA subsets in fibroblasts, identify HuR as an RBP implicated in the NO response (although HuR alone was insufficient for stabilizing several mRNAs by NO), and reveal that HO-1 induction by NO is regulated by HuR. |
Future plans: We will continue to investigate the turnover and translation of specific mRNAs that encode stress-response/proliferative proteins, to identify systematically the RBPs and microRNAs that regulate the expression of stress-response/proliferative proteins, and to study the functional consequences of miRNA/RBP interactions on target mRNAs. We are also interested in studying microscopically the subcellular localization of tagged mRNAs together with the RBPs/miRNAs that regulate their expression, and in developing efficient proteomic approaches to identify the collections of RBPs that associate with a given mRNA. Future work will also examine the mechanisms (transcriptional and post-transcriptional) that regulate microRNA expression levels, the function and target mRNAs for other RBPs of interest, and the subsets of mRNAs that are the targets of specific microRNAs of interest. Finally, we plan to study RBP expression systematically across tissues and ages using human tissue microarrays, as will continue to investigate the roles of RBPs in tumorigenesis. |
Selected publications since 2007: Abdelmohsen, K., Pullmann, R. Jr., Lal, A. Kim, H.H., Galban, S., Yang, X., Blethrow, J., Walker, M., Shubert, J., Gillespie, D.A., Furneaux, H., and Gorospe, M. Phosphorylation of HuR by Chk2 regulates SIRT1 expression. Mol. Cell 25: 543-57, 2007. Abdelmohsen, K., Lal, A., Kim, H. H., and Gorospe, M. Posttranscriptional Orchestration of an Anti-Apoptotic Program by HuR. Cell Cycle 15, 2007. Pullmann, R. Jr., Kim, H.H., Abdelmohsen, K., Lal, A., Martindale, J.L., Yang, X., and Gorospe, M. Analysis of Stability and Translation Regulatory RBP Expression Through Binding to Cognate mRNAs. Mol. Cell. Biol. 27: 6265-6278, 2007. Kim, H.S., Kuwano, Y., Zhan, M., Pullmann, R., Jr., Mazan-Mamczarz, K., Li, H., Kedersha, N., Anderson, P., Wilce, M. C. J., Gorospe, M.*,**, Wilce, J. A*. Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR. Mol. Cell. Biol., 27, 6806-6817, 2007. (*co-senior authors; **corresponding author). Xiao, L., Rao, J.N., Zou, T., Liu, L., Marasa, B.S., Chen, J., Turner, D.J., Zhou, H., Gorospe, M., and Wang, J.-Y. Polyamines Regulate the Stability of Activating Transcription Factor-2 mRNA through RNA-binding Protein HuR in Intestinal Epithelial Cells. Mol. Biol. Cell 18, 4579-4590, 2007. Zahn, J.M., Poosala, S., Owen, A.B., Ingram, D., Lustig, A., Carter, A., Weeraratna, A.T., Taub, D.D., Gorospe, M., Mazan-Mamczarz, K., Lakatta, E., Boheler, K.R., Xu, X., Mattson, M.P., Falco, G., Ko, M.S.H., Schlessinger, D., Firman, J., Kummerfeld, S.K., Wood, W., Zonderman, A.B., Kim, S. K., Becker, K.G. AGEMAP: a gene expression database for aging in mice. PloS Genetics, 3(11):e201, 2007. Lecona, E., Olmo, N., Turnay, J., Santiago-Gomez, A., Lopez de Silanes, I., Gorospe, M., and Lizarbe, M.A. Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms. Biochem. J. 409: 311-20, 2008. Zou, T., Liu, L., Rao, J.N., Marasa, B.S., Chen, J., Xiao, L., Zhou, H., Gorospe, M., Wang, J.-Y. Polyamines Modulate the Subcellular Localization of RNA-binding Protein HuR through AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin α1. Biochemical J. 409: 389-98, 2008. Galbán, S., Kuwano, Y., Pullmann, R. Jr., Martindale, J.L., Kim, H.H., Lal, A., Abdelmohsen, K., Yang, X., Dang, Y., Liu, J.O., Lewis, S.M., Holcik, M., and Gorospe, M. RNA-binding proteins HuR and PTB promote the translation of hypoxia-inducible factor 1α. Mol. Cell. Biol. 28: 93-107, 2008. Abdelmohsen, K., Kuwano, Y., Kim, H.H., Gorospe, M. Posttranscriptional Gene Regulation by RNA-Binding Proteins During Oxidative Stress: Implications for Cellular Senescence. Biol. Chem. 389: 243-55, 2008. M. Gorospe and R. de Cabo; AsSIRTing the DNA damage response. Trends in Cell Biology 18: 77-83, 2008. Casolaro, V., Srikantan, R.S., Gorospe, M., and Stellato, C. Posttranscriptional Regulation of IL-13 in T Cells: Role of the RNA-binding protein HuR. Journal of Allergy and Clinical Immunology 121: 853-9, 2008. Robinson, V.L., Shalhav, O., Otto, K., Kawai, T., Gorospe, M., and Rinker-Schaeffer, C.W. MAP kinase kinase 4 (MKK4) protein expression is subject to translational regulation in prostate cancer cell lines. Mol. Cancer Res. 6: 501-8, 2008. Lal. A., Kim H. H., Abdelmohsen, K., Kuwano, Y., Pullmann, R., Jr., Srikantan, S., Subrahmanyam, R, Martindale J.L., Yang, X., Ahmed, F., Navarro, F., Dykxhoorn, D, Lieberman, J, and Gorospe, M. p16INK4a Translation Suppressed by miR-24. PLoS ONE 3:e1864, 2008. Ishmael, F. T., Fang, X., Prabhu, V. V., Blackshear, P. J., Gorospe, M., Cheadle, C., and Stellato, C. Role of the RNA-binding Protein Tristetraprolin in Glucocorticoid-mediated Gene Regulation. J. Immunol. 180: 8342-53, 2008. Hucl, T., Rago, C., Gallmeier, E., Brody, J., Gorospe, M., and Kern, S. A syngeneic variance library for functional annotation of human variation: application to BRCA2. Cancer Research (In Press). Kim, H.H., Abdelmohsen, K., Lal, A., Pullmann, R. Jr., Yang, X., Galban, S., Srikantan, S., Martindale, J.L., Blethrow, J., Shokat, K.M.. and Gorospe, M. Nuclear Retention of HuR Through Phosphorylation by Cdk1. Genes & Development 22:1804-15, 2008. Kuwano, Y., Kim, H.H., Abdelmohsen, K., Pullmann, R. Jr., Martindale, J.L., Yang, X., and Gorospe, M. MKP-1 mRNA Stabilization and Translational Control by RNA-Binding Proteins HuR and NF90. Mol. Cell Biol 28:4562-75, 2008. van der Brug, M.P., Blackinton, J., Chandran, J., Hao, L.Y., Lal, A., Mazan-Mamczarz, K., Martindale, J.L., Xie, C., Ahmad, R., Thomas, K.J., Beilina, A., Gibbs, J.R., Ding, J., Myers, A.J., Zhan, M., Cai, H., Bonini, N.M., Gorospe, M., and M.R. Cookson. RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways. Proc. Natl. Acad. Sci. USA 105:10244-9, 2008. Kim, H.H and Gorospe, M.GU-Rich RNA: Expanding CUGBP1 Function, Broadening mRNA Turnover. Mol. Cell 29:151-2, 2008. Mazan-Mamczarz, K., Hagner, P.R., Corl, S., Wood W.H., Becker, K.G., Gorospe, M., Keene, J.D., A, Levenson, A.S., and Gartenhaus, R.B.: Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype. Oncogene 27:6151-63, 2008. Kuwano, Y., and Gorospe, M. Protecting the stress response, guarding the MKP-1 mRNA. Cell Cycle 7:2640-2, 2008. Kim, H.H., Gorospe, M. Phosphorylated HuR Shuttles in Cycles. Cell Cycle 7:3124-6, 2008. Kim, H.H., Yang, X., Kuwano, Y., and Gorospe, M. Modification at HuR(S242) Alters HuR Localization and Proliferative Influence. Cell Cycle 7:3371-7, 2008. Abdelmohsen, K., Srikantan, S., Kuwano, Y., and Gorospe, M. miR-519 Reduces Cell Proliferation by Lowering RNA-Binding Protein HuR Levels. Proc. Natl. Acad. Sci. USA 105:20297-302, 2008. Mazan-Mamczarz, M., Kuwano, Y., Zhan, M., White, E.J., Martindale, J.L., Lal, A., and Gorospe, M. Identification of a Signature Motif in Target mRNAs of RNA-Binding Protein AUF1. Nuc. Acids Res. (In Press, 2009). López de Silanes, I., Gorospe, M., Taniguchi, H., Abdelmohsen, K., Srikantan S., Alaminos, M., Berdasco, M., Urdinguio, R.J., Fraga, M.F., Jacinto, F.V., and Esteller, M. The RNA-binding protein HuR regulates DNA Methylation through stabilization of DNMT3b mRNA. Nuc. Acids Res. (2009, In press). Andersen, J.B., Mazan-Mamczarz, K., Zhan, M., Gorospe, M., Hassel, B.A. Ribosomal protein mRNAs are primary targets of regulation in RNase-L-induced senescence. RNA Biology (2009, In press). Kuwano, Y., Rabinovic, A., Srikantan, S., Gorospe, M.* (*Co-senior author), and Demple, B. Analysis of nitric oxide-stabilized mRNAs in human fibroblasts reveals HuR-dependent heme oxygenase-1 upregulation. Mol. Cell. Biol. (2009, In Press). Abdelmohsen, K., Srikantan, S., Yang, X., Lal, A., Kim, H.H., Kuwano, Y., Galban, S., Becker, K. G., Kamara, D., de Cabo, R., and Gorospe, M. Ubiquitin-mediated proteolysis of HuR by heat shock. EMBO J., (2009 In press). |
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