• Altered Expression of RNA-binding Proteins Modulates Tumor Progression by Re-programming Post-transcriptional Gene Regulatory Networks

      Brennan, Sarah Elizabeth; Wilson, Gerald M. (2010)
      AU-rich elements (AREs) are potent cis-acting determinants of mRNA decay and translational efficiency in mammalian cells. Since ARE-containing transcripts often encode factors that regulate cell division, apoptosis, angiogenesis, and inflammation, alterations in the metabolic fates of these mRNAs can profoundly influence cellular phenotypes associated with oncogenesis. We evaluated changes in the expression of four well characterized ARE-BPs (AUF1, TIA-1, HuR, and TTP) across a variety of human neoplastic syndromes using three principal methods: (i) cDNA arrays comparing expression in 154 tumors from 18 different tissue types versus patient-matched non-transformed tissues, (ii) meta-analyses of gene chip studies comparing expression in normal versus primary and metastatic tumors across diverse tissue types, and (iii) comparing EST and/or SAGE frequency between normal versus cancerous cells derived from many tissue sources. For three ARE-BPs surveyed; AUF1, TIA-1, and HuR, expression was not systematically dysregulated in cancers; however, in selected tissues mRNAs encoding these proteins were frequently up- or down-regulated to a significant extent. Conversely, TTP expression is repressed in many human tumors and in breast cancer correlates with increased expression of an angiogenic factor and is a negative prognostic indicator. In a cultured cancer cell model, restoring TTP expression slows cell proliferation, enhances sensitivity to selected pro-apoptotic stimuli, and decreases expression of vascular endothelial growth factor (VEGF) mRNA by accelerating the decay kinetics of this transcript. Finally, ribonome-wide screens for mRNAs that both associate with TTP and are regulated by TTP have identified a panel of TTP substrate transcripts that may be responsible for linking loss of TTP expression to the exacerbation of tumorigenic phenotypes. Together, these data indicate that the diminution of TTP expression observed in many cancers re-programs a post-transcriptional gene regulatory network, resulting in enhanced expression of several factors that may contribute to tumor aggressiveness.
    • Controlling LDL Receptor Expression by Regulating mRNA Decay Kinetics

      Vargas, Noelle; Wilson, Gerald M. (2009)
      Low density lipoprotein (LDL) is the most abundant cholesterol transport vehicle in plasma, and remains a major prognostic indicator of atherosclerotic risk. Hepatic LDL receptors are essential for limiting circulating LDL levels, since cholesterol internalized by the liver can be excreted via conjugation to bile salts. This relationship is clearly seen in familial hypercholesterolemic patients, where depletion or lack of functional LDL receptors dramatically increases circulating LDL, accelerating development of atherosclerosis and associated cardiovascular disease. The focus of this study is to characterize post-transcriptional mechanisms regulating hepatic expression of LDL receptors. In this study, we have found that TPA-induced stabilization of receptor mRNA is accompanied by activation of several mitogen activated protein kinase (MAPK) pathways in HepG2 cells. Initial screens with pharmacological inhibitors of specific MAPK pathways indicated that TPA-induced stabilization of LDL receptor mRNA requires the JNK, but not ERK or p38MAPK, pathways. An essential role for JNK activation in stabilization of receptor mRNA was further confirmed by siRNA knockdown experiments. Adenoviral transduction of a constitutively active mutant of MKK7, the JNK-activating kinase, further confirmed that JNK activation was sufficient to stabilize LDL receptor mRNA in the HepG2 model system. Finally, prolonged induction of JNK activity increased the steady-state levels of LDL receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. Perturbation of the hepatic JNK pathway may represent a novel anti-atherosclerotic therapeutic target, by enhancing the potential of liver cells to internalize and excrete plasma LDL cholesterol. In our efforts to identify the TPA responsive element within the LDL receptor mRNA, we demonstrated that multiple sequence elements within an Alu-rich region in the 3' untranslated region is responsible for TPA-induced stabilization. At present, no trans-acting factors have been identified that modulate mRNA decay kinetics through any of the Alu repetitive elements scattered throughout the human genome. However, preliminary findings described in this work supported by several observations reported in the literature raise the possibility that microRNA (miRNA)-mediated mechanisms might contribute to the regulated stability of LDL receptor mRNA through the Alu-rich region.
    • Post-transcriptional control of gene expression by the major inducible heat shock protein Hsp70

      Kishor, Aparna; Wilson, Gerald M. (2012)
      AU-rich elements (AREs) encoded within the 3'-untranslated regions (3'UTRs) of many mRNAs are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone, also interacts with ARE-like RNA substrates in vitro. The findings here define cellular roles for the interaction between Hsp70 and ARE-containing transcripts as well as mechanistic aspects of binding. Suppression of Hsp70 in cells destabilizes a reporter mRNA containing a high-affinity Hsp70 binding site. Consistent with these data, Hsp70 interacts with and stabilizes endogenous ARE-containing mRNAs encoding VEGF and Cox-2 in HeLa cells. Hsp70 recognition and stabilization of VEGF mRNA is mediated by an ARE-like sequence in the proximal 3'UTR. Stabilization of VEGF mRNA coincides with accumulation of Hsp70 protein levels in HL60 promyelocytic leukemia cells recovering from acute thermal stress. The minimum length of RNA recognized by Hsp70 is probably on the order of 30 nucleotides, which must be single-stranded. Hsp70 has no canonical RNA-binding motifs, but both the ATPase and peptide-binding domains, the two major structural modules of the protein, recognize and bind AREs with high specificity and mid-nanomolar affinity. The ATPase domain, however, is unable to restore VEGF transcript stability in HeLa cells where endogenous Hsp70 has been suppressed. In contrast, the peptide-binding domain is able to partially restore the half-life of this transcript. The ARE-directed mRNA stabilization and protein chaperone roles of Hsp70 are likely to be independent functions. This conclusion is supported by observations that the nucleotide cofactor-bound status of the ATPase domain does not impact the binding affinity between Hsp70 and an ARE-containing substrate, suggesting that the protein conformational changes associated with the chaperone cycle are unrelated to the interaction between Hsp70 and RNA. Additionally, stabilization of an endogenous mRNA target involves a mechanism that is unaffected by an inhibitor of Hsp70 chaperone function. Since constitutively elevated Hsp70 levels are a negative prognostic indicator for several types of cancer, we propose that Hsp70-directed stabilization of ARE-containing mRNAs that encode regulators of tumor growth and metastasis may provide a novel mechanism linking this protein to the development of aggressive neoplastic phenotypes.
    • Protein and RNA Determinants Regulating Binding of AUF1, A Major Post-Transcriptional Regulator of Gene Expression

      Zucconi, Beth E.; Wilson, Gerald M. (2013)
      AU-rich element RNA binding protein 1 (AUF1) regulates the stability of mRNAs containing AU-rich elements (AREs) in their 3'-untranslated regions. AREs are highly enriched in mRNAs that encode cytokines, cell cycle regulators, and other regulatory proteins, making AUF1 a critical factor in control of many cellular systems. Four AUF1 isoforms arise through alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggested that the different isoforms have varied functional and regulatory characteristics, but no detailed, quantitative analysis of their biochemical properties had been reported. Using purified recombinant forms of each AUF1 protein variant, we used chemical crosslinking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each isoform for ARE-containing RNA substrates and quantified binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. While all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, exon 2-encoded sequences inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization. Finally, fluorescence resonance energy transfer-based assays (FRET) showed that the different isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein (RNP) complexes, which likely constitute a mechanistic basis for differential function and regulation. Subsequently we evaluated RNA requirements for AUF1 binding. Despite a binding site size of 33-34 nucleotides on ARE substrates, p37<super>AUF1</super> requires only 15 nucleotides of AU-rich sequence to form stable RNPs in a larger RNA context. In particular, a 3'-guanine and 5'-base-specific sequence stabilize p37<super>AUF1</super> binding, but by a mechanism that does not involve new ionic contacts. However, van't Hoff and FRET analyses showed that AUF1 contacts with the 5&rsquo-extension contribute to RNA structural condensation. Reporter mRNAs containing minimal high-affinity target sequences associate with AUF1 by RNP-immunoprecipitation and are destabilized in an AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs, but also suggest that the role of AUF1 and proteins/miRNAs binding adjacent sites may be reciprocally regulated by local RNA structure.
    • Tristetraprolin functions as a tumor suppressor in metastatic breast cancer cells by a novel mechanism independent of mRNA binding

      Ross, Christina Ruth; Wilson, Gerald M. (2015)
      The RNA-binding protein tristetraprolin (TTP) targets and initiates decay of many mRNAs containing AU-rich elements. TTP expression is repressed across a wide range of human tumors and cancer cell lines relative to non-transformed tissues suggesting that TTP may function as a tumor suppressor in many cancers, and low TTP expression is a negative prognostic indicator in breast cancer. The range of pro-tumorigenic factors encoded by TTP-targeted mRNAs and the variety of neoplastic phenotypes that TTP has been reported to modulate in different cell types prompted the hypothesis that TTP may coordinately attenuate multiple tumor suppressive pathways in a single cell background. Here we show that TTP slows proliferation of the triple negative MDA-MB-231 breast cancer cell model by restricting passage through the G1/S checkpoint while concomitantly inhibiting stemness, cell migration, expression of select regulatory proteins including cyclin D1 and c-Myc, and tumor growth in murine xenograft models. However, suppression of cellular tumorigenic phenotypes and cell cycle markers was independent of TTP's canonical mRNA-destabilizing function, which is inactivated by constitutive ERK but not p38 MAP kinase signaling in MDA-MB-231 cells. Consistent with this model, a non-RNA-binding TTP mutant similarly suppressed MDA-MB-231 proliferation, stemness, and migration together with cyclin D1 and c-Myc levels. Protein turnover assays revealed that TTP expression accelerates the decay of cyclin D1 and c-Myc by a proteasome-dependent mechanism. Microarray analysis of MDA-MB-231 cells expressing TTP also revealed attenuated regulation of c-Myc target genes, however shRNA studies showed c-Myc knockdown in MDA-MB-231 cells does not result in the same attenuation of gene regulation, nor does loss of c-Myc phenocopy the effects of TTP expression. However, despite being dispensable for suppressing tumorigenic characteristics of MDA-MB-231 cells, the RNA-binding activity of TTP did induce a variety of specific gene expression changes despite limitations on its mRNA-destabilizing ability. Collectively our data establish a novel mechanism by which TTP can suppress several tumorigenic phenotypes and gene expression programs in a single cell model independently of its mRNA-destabilizing function. Given the diverse range of cancers where MAP kinase pathways are activated, these non-canonical TTP functions may be applicable to many tumor types.