Hu Antigen R Research Articles

The levels of the RNA binding protein Hu antigen R determine the druggability of the neddylation pathway in liver cancer

Hepatocellular carcinoma (HCC), the most common liver cancer, is an important leading cause of death worldwide. Neddylation is a post-translational modification involved in several processes such as cell growth, viability and development. Importantly, the neddylation pathway is upregulated in liver cancer and specifically enriched in patients with poor prognosis. Hu antigen R (HuR), is a RNA-binding protein that stabilizes target mRNAs involved in hepatocyte proliferation, differentiation and malignant transformation. And notably, HuR levels are highly representative in liver and colon cancer. A ground-breaking knowledge about HCC has been to identify that neddylation plays a role in HCC by regulating the liver oncogenic driver HuR. In addition, the neddylation inhibitor MLN4924 has shown antitumoral effects in vitro and in vivo in liver cancer, partly through HuR destabilization. Importantly, high levels of HuR made hepatoma cells more resistant to neddylation inhibition while low levels of HuR sensitized cells to the treatment, suggesting that the levels of HuR determine the druggability of the neddylation pathway in HCC. Overall, our findings highlight the impact that neddylation plays in liver cancer and open a completely new area of research, paving the way for novel therapeutical approaches.

Hu antigen R is required for NOX-1 but not NOX-4 regulation by inflammatory stimuli in vascular smooth muscle cells.

NOX-1 and NOX-4 are key enzymes responsible for reactive oxygen species (ROS) generation in vascular smooth muscle cells (VSMC). The RNA-binding protein Hu antigen R (HuR) is implicated in posttranscriptional regulation of gene expression; however, its role regulating NOX is unknown. We investigated transcriptional and posttranscriptional mechanisms underlying angiotensin II (AngII) and IL-1β regulation of NOX-1 and NOX-4 in VSMC and their implications in cell migration. Rat and human VSMC were stimulated with AngII (0.1 μmol/l) and/or IL-1β (10 ng/ml). NOX-1 and NOX-4 mRNA and protein levels, NOX-1 and NOX-4 promoter and 3'UTR activities, NADPH oxidase activity, ROS production, and cell migration were studied. IL-1β increased NOX-1 expression, NADPH oxidase activity and ROS production, and decreased NOX-4 expression and H2O2 production in VSMC. AngII potentiated the IL-1β-mediated induction of NOX-1 expression, NADPH oxidase activity, ROS production, and cell migration. However, AngII did not influence IL-1β-induced NOX-4 downregulation. AngII + IL-1β interfered with the decay of NOX-1 mRNA and promoted HuR binding to NOX-1 mRNA. Moreover, HuR blockade reduced NOX-1 mRNA stability and AngII + IL-1β-induced NOX-1 mRNA levels. IL-1β decreased NOX-4 expression through a transcriptional mechanism that involved response elements situated in the proximal promoter. AngII and/or IL-1β-induced cell migration were prevented by NOX-1 and HuR blockade and were augmented by NOX-4 overexpression. In VSMC HuR-mediated mRNA stabilization is partially responsible for AngII + IL-1β-dependent NOX-1 expression, whereas transcriptional mechanisms are involved in decreased NOX-4 expression induced by IL-1β. NOX4 and HuR regulation of NOX-1 contributes to VSMC migration, important in vascular inflammation and remodeling.

MiR-31 Functions as a Tumor Suppressor in Lung Adenocarcinoma Mainly by Targeting HuR.

Gene expression is widely regulated by miRNAs and RNA binding proteins. In this study, we mainly focused on miR-31 and a RNA binding protein, HuR (Hu antigen R). The levels of miR-31 and HuR in lung carcinoma cells and lung cancer tissues were explored using RT-qPCR and western blot, respectively. Luciferase reporter assay was used to determine the target gene of miR-31. Cell apoptosis and migration were studied using flow cytometry and the transwell invasion assay. The down-stream genes of HuR were explored with western blot assay. miR-31 was decreased in lung carcinoma cells and lung cancer tissues, while the protein level of HuR was increased. HuR was the target gene of miR-31. Inhibition of miR-31 and overexpression of HuR resulted in the upregulation of cyclins A2, B1, D1 and VEGF (vascular endothelial growth factor). Furthermore, overexpression of miR-31 prompted lung cancer cell apoptosis and inhibited cell migration. Reduction of miR-31 expression enhanced lung cancer proliferation and migration by repressing HuR expression.

CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.

Abstract 2449: Targeting an “undruggable” RNA-binding protein: Discovery of small molecule inhibitors of HuR for novel breast cancer therapy

Abstract Post-transcriptional gene regulation is essential for normal development, but when dysregulated, has many implications in disease conditions, including cancer. The RNA-binding proteins (RBPs) are critical trans factors that associate with specific cis elements present in mRNAs, thereby regulating the fate of target mRNAs. Due to the lack of well-defined binding pocket, RBPs have been considered “undruggable targets”. The RBP Hu antigen R (HuR) is overexpressed in many types of cancer, including breast cancer. Elevated cytoplasmic HuR level correlates with high-grade malignancy and serves as a prognostic factor of poor clinical outcome in breast cancer. HuR promotes tumorigenesis by interacting with cancer-associated mRNAs, those mRNAs encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. HuR also modulates the sensitivity of breast cancer cells to chemotherapy. Our hypothesis is that small molecule compounds that disrupt the HuR-mRNA interaction will block HuR function, leading to the decay and reduced translation of mRNAs of the target genes critical for breast cancer cell growth and progression. High throughput screening (HTS) was carried out in several chemical libraries (∼23,000 compounds) using fluorescence polarization (FP) assay and identified a series of initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit ST-3 and its optimized analogs, specifically shortened HuR target mRNAs’ half-life and decreased the level of the encoded proteins (Bcl-2, XIAP and Msi1/2). Moreover, those compounds selectively inhibited breast cancer cell proliferation but not normal cells. Knocking down HuR in breast cancer cells attenuated the activity of those HuR-mRNA disruptors. More cell-based assays are carrying out to delineate the mechanism of action. We are also testing the antitumor efficacy of those HuR inhibitors in mouse xenograft models. In conclusion, we identified potential small molecule disrupters of HuR-mRNA interaction for potential novel cancer therapy that inhibited breast cancer with HuR overexpression. Citation Format: Xiaoqing Wu, Lan Lan, Amber Smith, Rebecca Marquez, David Wilson, Steven Rogers, Philip Gao, Scott Lovell, John Karanicolas, Dan Dixon, Jeffrey Aubé, Liang Xu. Targeting an “undruggable” RNA-binding protein: Discovery of small molecule inhibitors of HuR for novel breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2015-2449

HuR keeps interferon-β mRNA stable.

The expression of interferon (IFN) β is highly induced in immune and nonimmune cells in response to virus infection. This upregulation is mediated at both transcriptional and posttranscriptional levels. Whereas the signaling pathways triggered by innate virus receptors leading to transcription factor activation have been extensively studied, the mechanisms by which IFN-β mRNA stability is posttranscriptionally controlled are not fully understood. In this issue of the European Journal of Immunology, Herdy et al. [Eur. J. Immunol. 2015 45:1500-1511] show that a human RNA-binding protein, embryonic lethal, abnormal vision, drosophila-like 1 (ELAVL1)/Hu antigen R (HuR), strongly associates with IFN-β mRNA via AU-rich sequences present in its 3' untranslated region in various human cell lines. The authors show that ELAVL1/HuR is required for the stabilization of IFN-β mRNA, and suppression of ELAVL1/HuR by its inhibitor MS-444 leads to impaired expression of IFN-β in response to viral dsRNA treatment. Thus, this study uncovers a novel mechanism of posttranscriptional IFN-β mRNA regulation in response to virus infection, through IFN-β stabilization by ELAVL1/HuR. Future studies are expected to identify further regulatory mechanisms of IFN-β stabilization and destabilization in the course of antiviral responses.

MAP kinase phosphatase-1 expression is regulated by 15-deoxy-Δ12,14-prostaglandin J2 via a HuR-dependent post-transcriptional mechanism

In the present study, we demonstrate a mechanism through which 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces MKP-1 expression in rat primary astrocytes, leading to the regulation of inflammatory responses. We show that 15d-PGJ2 enhances the efficiency of MKP-1 pre-mRNA processing (constitutive splicing and 3′-end processing) and increases the stability of the mature mRNA. We further report that this occurs via the RNA-binding protein, Hu antigen R (HuR). Our experiments show that HuR knockdown abrogates the 15d-PGJ2-induced increases in the pre-mRNA processing and mature mRNA stability of MKP-1, whereas HuR overexpression further enhances the 15d-PGJ2-induced increases in these parameters. Using cysteine (Cys)-mutated HuR proteins, we show that the Cys-245 residue of HuR (but not Cys-13 or Cys-284) is critical for the direct binding of HuR with 15d-PGJ2 and the effects downstream of this interaction. Collectively, our data show that HuR is a novel target of 15d-PGJ2 and reveal HuR-mediated pre-mRNA processing and mature mRNA stabilization as important regulatory steps in the 15d-PGJ2-induced expression of MKP-1. The potential to use a small molecule such as 15d-PGJ2 to regulate the induction of MKP-1 at multiple levels of gene expression could be exploited as a novel therapeutic strategy aimed at combating a diverse range of MKP-1-associated pathologies.

NEDDylation in liver cancer: The regulation of the RNA binding protein Hu antigen R.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer death. The current view of cancer progression and malignancy supports the notion that cancer cells must undergo through a post-translational modification (PTM) regulation and a metabolic switch or reprogramming in order to progress in an unfriendly environment. NEDDylation is a post-translational modification of the proteins involved in several processes such as cell growth, viability and development. A ground-breaking knowledge on a new critical aspect of HCC research has been to identify that NEDDylation plays a role in HCC by regulating the liver oncogenic driver Hu antigen R (HuR). HuR is a RNA-binding protein that stabilizes target mRNAs involved in cell dedifferentiation, proliferation, and survival, all well-established hallmarks of cancer. And importantly, HuR levels were found to be highly representative in liver and colon cancer. These findings open a completely new area of research, exploring the impact that NEDDylation plays in liver diseases and paving the way for novel therapeutical approaches.

The RNA-binding protein HuR/ELAVL1 regulates IFN-βmRNA abundance and the type I IFN response.

Secretion of type I interferon (IFN) is the first cellular reaction to invading pathogens. Despite the protective function of these cytokines, an excessive response to their action can contribute to serious pathologies, such as autoimmune diseases. Transcripts of most cytokines contain adenylate-uridylate (A/U)-rich elements (AREs) that make them highly unstable. RNA-binding proteins (RBPs) are mediators of the regulatory mechanisms that determine the fate of mRNAs containing AREs. Here, we applied an affinity proteomic approach and identified lethal, abnormal vision, drosophila-like 1 (ELAVL1)/Hu antigen R (HuR) as the predominant RBP of the IFN-β mRNA ARE. Reduced expression or chemical inhibition of HuR severely hampered the type I IFN response in various cell lines and fibroblast-like synoviocytes isolated from joints of rheumatoid arthritis patients. These results define a role for HuR as a potent modulator of the type I IFN response. Taken together, HuR could be used as therapeutic target for diseases where type I IFN production is exaggerated.

HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

Hu Antigen R (HuR) Is a Positive Regulator of the RNA-binding Proteins TDP-43 and FUS/TLS: Implications for Amyotrophic Lateral Sclerosis

Posttranscriptional gene regulation is governed by a network of RNA-binding proteins (RBPs) that interact with regulatory elements in the mRNA to modulate multiple molecular processes, including splicing, RNA transport, RNA stability, and translation. Mounting evidence indicates that there is a hierarchy within this network whereby certain RBPs cross-regulate other RBPs to coordinate gene expression. HuR, an RNA-binding protein we linked previously to aberrant VEGF mRNA metabolism in models of SOD1-associated amyotrophic lateral sclerosis, has been identified as being high up in this hierarchy, serving as a regulator of RNA regulators. Here we investigated the role of HuR in regulating two RBPs, TDP-43 and FUS/TLS, that have been linked genetically to amyotrophic lateral sclerosis. We found that HuR promotes the expression of both RBPs in primary astrocytes and U251 cells under normal and stressed (hypoxic) conditions. For TDP-43, we found that HuR binds to the 3' untranslated region (UTR) and regulates its expression through translational efficiency rather than RNA stability. With HuR knockdown, there was a shift of TDP-43 and FUS mRNAs away from polysomes, consistent with translational silencing. The TDP-43 splicing function was attenuated upon HuR knockdown and could be rescued by ectopic TDP-43 lacking the 3' UTR regulatory elements. Finally, conditioned medium from astrocytes in which HuR or TDP-43 was knocked down produced significant motor neuron and cortical neuron toxicity in vitro. These findings indicate that HuR regulates TDP-43 and FUS/TLS expression and that loss of HuR-mediated RNA processing in astrocytes can alter the molecular and cellular landscape to produce a toxic phenotype.

The Clinical and Pathological Significance of Cyclooxygenase-2 (Cox-2) and Human Antigen Receptor (Hur) Expression in Non Small Cell Lung Cancers

ABSTRACT Aim: Cyclooxygenase-2 (COX-2) is implicated in tumor proliferation, angiogenesis, tumor invasiveness, cell-mediated immunity and resistance to apoptosis. Hu-antigen R (HuR) is also considered to play a central role in tumor formation, growth and metastasis by binding to mRNAs encoding proteins such as COX-2. The present study aimed to evaluate the clinical significance of COX-2 and HuR protein expression in non-small cell lung cancers (NSCLC). Methods: COX-2 and HuR expression was assessed immunohistochemically on tissue microarrays of 81 surgically resected NSCLC and was analyzed in relation with clinicopathological characteristics and patients' survival. Results: Enhanced COX-2 expression was significantly associated with the presence of lymphovascular invasion and increased tumor proliferative capacity (p=0.031 and p=0.023, respectively). Enhanced total HuR expression was significantly associated with tumour histopathological type and presence of lymph node metastases, as well as, with increased tumour proliferative capacity and poor patients' outcome (p=0.039, p=0.017, p=0.033 and p=0.022, respectively). Concomitant elevated expression levels of HuR and COX-2 were significantly associated with tumour histopathological type and increased tumor proliferative capacity (p=0.002 and p=0.045, respectively). Enhanced total HuR expression was significantly associated with increased COX-2 expression (p=0.015). Conclusions: The present study supported evidence that COX-2 and HuR may have a role in the malignant transformation and growth of NSCLC. Particularly, COX-2 and HuR could be considered as potential biomarkers indicating tumor aggressiveness. Nevertheless, more studies are needed to elucidate their clinical significance. Disclosure: All authors have declared no conflicts of interest.

Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.

Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

A single element in the 3'UTR of the apical sodium-dependent bile acid transporter controls both stabilization and destabilization of mRNA.

mRNA stability appears to play a key role in the ontogenic regulation of the apical sodium-dependent bile acid transporter (ASBT). The RNA-binding proteins Hu antigen R (HuR) and tristetraprolin (TTP) stabilize and destabilize ASBT mRNA, respectively. Potential HuR-binding sites were assessed by sequence analysis in the context of prior invitro functional analyses of the rat ASBT 3'UTR. Wild-type and mutant-binding sites were investigated by gel-shift analysis using IEC-6 cell extracts. The functional consequences of binding site mutations were assessed using two different hybrid reporter constructs linking the 3'UTR element to either a luciferase or a β-globin coding mRNA sequence. A specific metastasis-associated gene 1 (MTA1) cis-element was identified in the ASBT 3'UTR that became associated with proteins in IEC-6 cell extracts and could be supershifted by anti-HuR or anti-TTP antibodies. Mutation of this cis-element abrogated the gel shift of IEC-6 proteins. Furthermore, hybrid constructs containing a mutant MTA1 element had reduced responses to modulation of HuR or TTP. For the first time, we have identified a single specific sequence element in the 3'UTR of the rat ASBT mRNA that mediates counter-regulatory changes in mRNA abundance in response to both HuR and TTP.

Clinical significance of HuR expression in human malignancy.

Hu-antigen R (HuR) is an RNA-binding protein that regulates the stability, translation, and nucleus-to-cytoplasm translocation of target mRNAs. The aim of the present review was to summarize and present the currently available information in the English literature on HuR expression in various human tumors, verifying its possible clinical significance. HuR function is directly linked to its subcellular localization. In normal cells, HuR is mostly localized in the nucleus, while in malignant cells, an increase in cytoplasmic HuR levels has been noted, in both cell lines and tissue samples. Moreover, in malignancy, elevated HuR expression levels and cytoplasmic immunohistochemical pattern have been correlated with advanced clinicopathological parameters and altered expression levels of proteins implicated in neoplasia. Additionally, elevated HuR expression levels and mainly cytoplasmic immunohistochemical pattern were correlated with decreased patients' survival rate in various human tumors. HuR is a putative drug target for cancer therapy, since it is expressed ubiquitously in malignant clinical samples and has an apparently consistent role in tumor formation and progression.

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

The structure of the ARE-binding domains of Hu antigen R (HuR) undergoes conformational changes during RNA binding

Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Å resolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Å resolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR.

Post-transcriptional Regulation of Meprin α by the RNA-binding Proteins Hu Antigen R (HuR) and Tristetraprolin (TTP)

Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3'-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease.

Tissue-specific control of brain-enriched miR-7 biogenesis

MicroRNA (miRNA) biogenesis is a highly regulated process in eukaryotic cells. Several mature miRNAs exhibit a tissue-specific pattern of expression without an apparent tissue-specific pattern for their corresponding primary transcripts. This discrepancy is suggestive of post-transcriptional regulation of miRNA abundance. Here, we demonstrate that the brain-enriched expression of miR-7, which is processed from the ubiquitous hnRNP K pre-mRNA transcript, is achieved by inhibition of its biogenesis in nonbrain cells in both human and mouse systems. Using stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry combined with RNase-assisted RNA pull-down, we identified Musashi homolog 2 (MSI2) and Hu antigen R (HuR) proteins as inhibitors of miR-7 processing in nonneural cells. This is achieved through HuR-mediated binding of MSI2 to the conserved terminal loop of pri-miR-7. Footprinting and electrophoretic gel mobility shift analysis (EMSA) provide further evidence for a direct interaction between pri-miR-7-1 and the HuR/MSI2 complex, resulting in stabilization of the pri-miR-7-1 structure. We also confirmed the physiological relevance of this inhibitory mechanism in a neuronal differentiation system using human SH-SY5Y cells. Finally, we show elevated levels of miR-7 in selected tissues from MSI2 knockout (KO) mice without apparent changes in the abundance of the pri-miR-7 transcript. Altogether, our data provide the first insight into the regulation of brain-enriched miRNA processing by defined tissue-specific factors.