| RNA Regulation Section |
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Myriam Gorospe, Ph.D., Chief
Senior Investigator |
| Overview |
| Research Summary: Aging is characterized by a general decline in the ability of individuals to adequately respond to different stresses, either environmental or endogenously generated. Changes in the expression of many stress-regulated genes is believed to play an important role in determining cell fate. While the transcriptional events serving to regulate the expression of these genes have been extensively studied, it is becoming increasingly clear that post-transcriptional regulatory mechanisms also play a critical role regulating gene expression during stress. These post-transcriptional processes, still poorly understood, rely primarily on changes in the stability of the mRNA, but are likewise linked to events such as nuclear export of mRNA and mRNA translation, which are linked to mRNA turnover. Our long-term efforts are three-fold: 1) to search for RNA-binding proteins and target mRNA regions involved in regulating the export, stability and translation of labile mRNAs; 2) to elucidate the signaling events that regulate such post-transcriptional modifications; and 3) to study the implications of post-transcriptional gene regulation on physiological and pathological processes. |
| Regulation of Stress-Response Genes Through Altered mRNA Turnover: We and others have shown that the expression of specific stress-response genes (p21, cyclin D1, etc.) is highly influenced by changes in the stability of the encoding mRNAs. Major research efforts underway in the laboratory seek to carry out global assessments of changes in mRNA turnover using cDNA arrays. We previously developed a new method based on the use of cDNA arrays to investigate the global contribution of transcription and mRNA turnover and applied it to the study of stress-regulated gene expression patterns. This novel method compares large-scale hybridization patterns generated with steady-state mRNA with those generated using newly transcribed RNA, generated and isolated using the nuclear run-on technology. By comparing the values from each analysis we can ascertain the extent to which stimulus-regulated modifications in gene expression are due to transcriptional changes and to post-transcriptional changes. In the stress study, changes in mRNA stability were found to account for approximately 50% of all gene expression changes. We are actively applying this methodology to the large-scale investigation of mRNA turnover changes during other cellular responses. |
| Signaling Events Regulating mRNA Turnover in Response to Stress: Several recent studies have provided increasing support for the notion that mRNA stability is regulated through mechanisms akin to those controlling gene transcription, i.e., signal transduction pathways involving phosphorylation events. While transport of the RNA-binding protein HuR from the nucleus to the cytoplasm is emerging as a key step in the activation of HuR function, the mechanisms underlying this process remain poorly understood. We recently identified the AMP-activated kinase (AMPK), an enzyme involved in responding to metabolic stresses, as a potent regulator of the levels of cytoplasmic HuR. Inhibition of AMPK increased HuR presence in the cytoplasm, enhanced binding of HuR to mRNAs encoding p21, cyclin B1 and cyclin A, and elevated their expression and half-life. Conversely, AMPK activation resulted in reduced cytoplasmic HuR, decreased levels and half-life of mRNAs encoding p21, cyclin A and cyclin B1, and diminished HuR association with the corresponding transcripts. We thus propose a novel function for AMPK as a regulator of cytoplasmic HuR levels, which in turn influences the mRNA-stabilizing function of HuR and the expression of HuR target transcripts. Among the specific target molecules that appear to mediate the transport of HuR through the nuclear pore are several import proteins, including importin a1, importin b1 and importin b2. Ongoing studies are aimed at elucidating the precise mechanisms whereby AMPK regulates HuR import through the various importins. Additional signal transduction pathways involved in regulating mRNA turnover are the subject of ongoing investigation. |
| Role of HuR on Protein Translation: HuR is a ubiquitously expressed member of the Hu family of RNA-binding proteins, which also comprises the primarily neuronal proteins HuB, HuC, and HuD. Hu proteins possess three RNA-recognition motifs through which they bind with high affinity and specificity to target mRNAs containing regions rich in adenines and uracils (AU-rich elements or AREs), and to regulate their stability, translation, or both. Our recent findings provide evidence in support of an additional level of post-transcriptional gene regulation by HuR in response to genotoxins. Exposure to UVC strongly induced expression of the tumor suppressor p53 without elevating p53 mRNA levels or the cytoplasmic export of the p53 mRNA. Instead, UVC irradiation strongly promoted p53 translation. HuR was found to associate with the 3'-untranslated region (UTR) of the p53 mRNA in a UVC-dependent manner in vitro and in vivo and to enhance p53 translation after UVC. Our current efforts are focused on elucidating whether other HuR target mRNAs might also be subject to altered translation, and testing whether HuR may jointly increase the stability and translation of target mRNAs during specific cellular responses. |
| Regulation of mRNA Turnover During Cellular Senescence: Cellular aging is accompanied by alterations in gene expression patterns. Using three models of replicative senescence, we recently described the influence of HuR in coordinately regulating the expression of cyclin A, cyclin B1 and c-fos, whose expression decreases during senescence. We demonstrated that HuR levels, HuR binding to target mRNAs encoding cyclin A, cyclin B1 and c-fos, and the half-lives of such mRNAs, were lower in senescent cells. We further showed that HuR levels directly influenced the senescent phenotype and that mRNA turnover played a critical role during the process of replicative senescence. Given that the cytoplasmic presence of HuR (believed to be required for HuR function) was recently shown to be inhibited by AMPK, we have been examining the function of this kinase during cellular senescence. We recently found that AMP:ATP ratios are higher in senescent cells, and AMPK activity was accordingly elevated in senescent cells. Current work in the laboratory is investigating the involvement of AMPK and HuR in the process of in vitro senescence. Experimental approaches based on the use of chemical regulators of AMPK activity as well as adenoviral vectors expressing either constitutively active or dominant negative isoforms of the kinase, were found to directly influence the implementation of the senescent phenotype. The bulk of our results indicate that AMPK activation can cause premature fibroblast senescence, since interventions that induced AMPK activity cause premature senescence in IDH4 and IMR-90 cells, while reductions in AMPK activity cause delays in the onset of cellular senescence. We further propose that AMPK-triggered cellular senescence is implemented, at least in part, through an AMPK-triggered reduction in HuR function. |
| Although the link between in vitro cellular senescence and human aging remains controversial, a diminution in proliferative capacity is also a hallmark of in vivo aging. Therefore, knowledge of the mechanisms regulating gene expression during in vitro senescence is likely to aid in our understanding of in vivo aging, as well as contribute to our comprehension of age-related diseases such as cancer and hyperplasia, where control of proliferation is lost. Our findings further suggest that orchestrated gene expression during senescence may be regulated by proteins such as HuR that coordinately regulate the stability of mRNAs encoding critical proliferation- and senescence-associated proteins. The study of additional senescence-associated labile mRNAs and RNA-binding proteins has become an area of great interest within the Unit. |
| Influence of the von Hippel-Lindau (VHL) Tumor Suppressor on Gene Expression: The precise mechanisms whereby the VHL gene product exerts its tumor suppressor function have not been fully elucidated. Based on evidence that the von Hippel-Lindau (VHL) tumor suppressor protein is associated with polysomes and interacts with translation regulatory factors, we recently set out to investigate the potential influence of pVHL on protein translation. To this end, renal cell carcinoma (RCC) cells that either lacked or expressed VHL through stable transfection were used to prepare RNA from cytosolic and polysome-bound fractions. Hybridization of cDNA arrays using RNA from each fraction revealed an influence of pVHL on protein translation: a subset of transcripts preferentially associated with polysomes in VHL-deficient cells (revealing genes that were subject to pVHL-repressed translation) and a subset of transcripts preferentially associated with polysomes in VHL-expressing cells (genes subject to pVHL-induced translation). Among the transcripts that were preferentially associated with polysomes in VHL-deficient cells was that encoding the tumor necrosis factor (TNF)a, an observation that further shown to be mediated through the TNFa 3' UTR. Among the transcripts that were preferentially associated with polysomes in VHL-expressing cells was the mRNA encoding the tumor suppressor p53. Additional assays revealed that the RNA-binding protein HuR was capable of binding the 3’UTR of the p53 mRNA preferentially in VHL-expressing cells. Use of siRNA effectively reduced HuR expression and markedly decreased p53 translation and p53 abundance. Our ongoing efforts demonstrate a novel function for the pVHL tumor suppressor as regulator of protein translation. Given the well established links of TNFa and p53 on tumor development, our findings further suggest that VHL-mediated changes in protein translation may contribute to pVHL’s tumor suppressive functions in RCC. |
