Dominique Morello

c-myc Internal Ribosome Entry Site Activity Is Developmentally Controlled and Subjected to a Strong Translational Repression in Adult Transgenic Mice

ABSTRACT The expression of c-myc proto-oncogene, a key regulator of cell proliferation and apoptosis, is controlled at different transcriptional and posttranscriptional levels. In particular, the c-myc mRNA contains an internal ribosome entry site (IRES) able to promote translation initiation independently from the classical cap-dependent mechanism. We analyzed the variations of c-myc IRES activity ex vivo in different proliferating cell types, and in vivo in transgenic mice expressing a bicistronic dual luciferase construct. c-myc IRES efficiency was compared to that of encephalomyocarditis virus (EMCV) IRES under the same conditions. The c-myc IRES was active but with variable efficiency in all transiently transfected cell types; it was also active in the 11-day- old (E11) embryo and in some tissues of the E16 embryo. Strikingly, its activity was undetected or very low in all adult organs tested. In contrast, EMCV IRES was very active in most cell types ex vivo, as well as in embryonic and adult tissues. These data suggest a crucial role of IRES in the control of c-mycgene expression throughout development, either during embryogenesis where its activity might participate in cell proliferation or later on, where its silencing could contribute to the downregulation of c-myc expression, whose deregulation leads to tumor formation.

Synchronous and regulated expression of two AU-binding proteins, AUF1 and HuR, throughout murine development.

The AUF1 (hnRNPD) and HuR (ELAV-like) proteins, potential trans-acting factors for regulated mRNA decay, bind in vitro to A+U-rich elements (AREs) found in the 3′ untranslated region (3′ UTR) of many labile transcripts. In an effort to determine whether these trans-acting factors are likely to play a role in embryogenesis, we have analysed their expression during mouse development both at the mRNA and protein levels. We show that AUF1 and HuR are expressed at all the developmental stages analysed from day 8.5 of embryonic development to adulthood. Expression levels are dynamic, varying between tissues and developmental stages. However, a strong positive correlation between AUF1 and HuR protein levels was observed in all examined tissues. Finally, we compared AUF1 and HuR expression with accumulation of one common target mRNA, c-myc. The similar spatio-temporal distribution of these proteins and of c-myc mRNA is in agreement with a potential concerted role in ARE-mediated control of mRNA stability.

Temporally regulated traffic of HuR and its associated ARE-containing mRNAs from the chromatoid body to polysomes during mouse spermatogenesis.

Background In mammals, a temporal disconnection between mRNA transcription and protein synthesis occurs during late steps of germ cell differentiation, in contrast to most somatic tissues where transcription and translation are closely linked. Indeed, during late stages of spermatogenesis, protein synthesis relies on the appropriate storage of translationally inactive mRNAs in transcriptionally silent spermatids. The factors and cellular compartments regulating mRNA storage and the timing of their translation are still poorly understood. The chromatoid body (CB), that shares components with the P. bodies found in somatic cells, has recently been proposed to be a site of mRNA processing. Here, we describe a new component of the CB, the RNA binding protein HuR, known in somatic cells to control the stability/translation of AU-rich containing mRNAs (ARE-mRNAs). Methodology/Principal Findings Using a combination of cell imagery and sucrose gradient fractionation, we show that HuR localization is highly dynamic during spermatid differentiation. First, in early round spermatids, HuR colocalizes with the Mouse Vasa Homolog, MVH, a marker of the CB. As spermatids differentiate, HuR exits the CB and concomitantly associates with polysomes. Using computational analyses, we identified two testis ARE-containing mRNAs, Brd2 and GCNF that are bound by HuR and MVH. We show that these target ARE-mRNAs follow HuR trafficking, accumulating successively in the CB, where they are translationally silent, and in polysomes during spermatid differentiation. Conclusions/Significance Our results reveal a temporal regulation of HuR trafficking together with its target mRNAs from the CB to polysomes as spermatids differentiate. They strongly suggest that through the transport of ARE-mRNAs from the CB to polysomes, HuR controls the appropriate timing of ARE-mRNA translation. HuR might represent a major post-transcriptional regulator, by promoting mRNA storage and then translation, during male germ cell differentiation.

HuR-mediated control of C/EBPbeta mRNA stability and translation in ALK-positive anaplastic large cell lymphomas.

The CCAAT/enhancer-binding protein β (C/EBPβ) plays a major role in the pathogenesis of anaplastic large cell lymphomas (ALCL) that express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) tyrosine kinase (ALK+). Although ALK-mediated C/EBPβ transcriptional activation has been reported, C/EBPβ mRNA possesses U- and AU-rich domains in its 3′-untranslated region (3′-UTR) that might be privileged targets for posttranscriptional control in ALK+ ALCLs. The purpose of this study was to explore this possibility. By using human ALCL-derived cells and a murine model of ALK-transformed cells, we show that the AU-binding protein HuR binds to the 3′-UTR of C/EBPβ mRNA, as previously reported in adipocytes, and that NPM-ALK enhances this interaction. Interaction between HuR and C/EBPβ mRNA impacts on C/EBPβ gene expression at both the mRNA and protein levels. Indeed, C/EBPβ mRNA stability following HuR silencing is reduced and reaches the value observed in ALK-inactivated cells. Remarkably, HuR expression is not modified by NPM-ALK, but its association with actively translating polysomes is dramatically increased in ALK+ cells. HuR/polysomes association diminishes when NPM-ALK activity is inhibited and is accompanied by a concomitant decrease of C/EBPβ mRNA translation. Finally, we show that HuR and NPM-ALK colocalized in cytoplasmic granules and HuR is phosphroylated on tyrosine residues in ALK+ ALCL cells. Our study thus demonstrates that C/EBPβ is indeed regulated at the posttranscriptional level by HuR in ALK+ cells, leading us to propose that part of NPM-ALK oncogenic properties relies on its ability to modify HuR properties in the cytoplasm and hence to alter expression of key actors of transformation. Mol Cancer Res; 9(4); 485–96. ©2011 AACR.

THE RNA-BINDING PROTEIN ELAVL1/HUR IS ESSENTIAL FOR MOUSE SPERMATOGENESIS, ACTING BOTH AT MEIOTIC AND POSTMEIOTIC STAGES

Posttranscriptional mechanisms are crucial to regulate spermatogenesis. Accurate protein synthesis during germ cell development relies on RNA binding proteins that control the storage, stability, and translation of mRNAs in a tightly and temporally regulated manner. Here, we focused on the RNA binding protein Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) known to be a key regulator of posttranscriptional regulation in somatic cells but the function of which during gametogenesis has never been investigated. In this study, we have used conditional loss- and gain-of-function approaches to address this issue in mice. We show that targeted deletion of HuR specifically in germ cells leads to male but not female sterility. Mutant males are azoospermic because of the extensive death of spermatocytes at meiotic divisions and failure of spermatid elongation. The latter defect is also observed upon HuR overexpression. To elucidate further the molecular mechanisms underlying spermatogenesis defects in HuR-deleted and -overexpressing testes, we undertook a target gene approach and discovered that heat shock protein (HSP)A2/HSP70-2, a crucial regulator of spermatogenesis, was down-regulated in both situations. HuR specifically binds hspa2 mRNA and controls its expression at the translational level in germ cells. Our study provides the first genetic evidence of HuR involvement during spermatogenesis and reveals Hspa2 as a target for HuR.

AU-Rich Elements Regulate Drosophila Gene Expression

ABSTRACT In mammals, AU-rich elements (AREs) are critical regulators of mRNA turnover. They recruit ARE-binding proteins that inhibit or stimulate rapid mRNA degradation in response to stress or developmental cues. Using a bioinformatics approach, we have identified AREs in Drosophila melanogaster 3′ untranslated regions and validated their cross-species conservation in distant Drosophila genomes. We have generated a Drosophila ARE database (D-ARED) and established that about 16% of D. melanogaster genes contain the mammalian ARE signature, an AUUUA pentamer in an A/U-rich context. Using candidate ARE genes, we show that Drosophila AREs stimulate reporter mRNA decay in cultured cells and in the physiological context of the immune response in D. melanogaster. In addition, we found that the conserved ARE-binding protein Tis11 regulates temporal gene expression through ARE-mediated decay (AMD) in D. melanogaster. Our work reveals that AREs are conserved and functional cis regulators of mRNA decay in Drosophila and highlights this organism as a novel model system to unravel in vivo the contribution of AMD to various processes.

lacZ and ubiquitously expressed genes: should divorce be pronounced?

Since the demonstration that bacterial β-galactosidase activity can be visualised in mammalian cells by simple and sensitive methods, the Escherichia coli lacZgene has been employed in various types of studies. For example, its use as a reporter gene has been very beneficial to the analysis of cis-acting regulatory sequences using transgenic mice, in particular those concerning genes expressed during embryonic development. More recently, gene-trap and ‘knock-in’ studies have shown that, in these systems as well, lacZ expression can faithfully report the activity of a gene it is fused with (see, for example, Colucci-Guyon et al., 1994). However, while this holds true for a vast majority of cases, several groups have reported that lacZ sequences could have a silencing effect on a certain category of fusion genes. A striking example of such an effect has come from the analysis ofH-2K cis-acting regulatory sequences (Cohen-Tannoudji et al., 2000). The H-2K gene codes for a major histocompatibility complex class I antigen that is expressed at the surface of most cells of adult mice. In order to dissect the H-2K gene regulatory sequences, transgenic mice containing different H-2K/lacZ constructs were derived. None of the 31 transient transgenics or permanent lines appropriately expressed the transgene. Instead, the H-2K/lacZtransgenes were either transcriptionally silent or behaved as an enhancer trap construct, that is, were expressed differently in every transgenic mouse line. This was not due to the lack of appropriate regulatory sequences in the H-2K/lacZ constructs because, when the very sameH-2K genomic sequences were linked to various eukaryotic coding sequences such as those of the hGH,

Stabilization of Dll1 mRNA by Elavl1/HuR in neuroepithelial cells undergoing mitosis

Delta/Notch-dependent lateral inhibition becomes enhanced in neuroepithelial cells undergoing mitosis. We report that the RNA-binding protein Elavl1/HuR localizes within the cytoplasm of these cells, interacts with the 3′-untranslated region of Dll1, and stabilizes transcripts containing this sequence, thus favoring Notch signaling.

Impaired gametogenesis in mice that overexpress the RNA‐binding protein HuR

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA‐binding protein HuR increases the half‐life of some messenger RNAs that contain adenylate/uridylate‐rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes. This work provides the first evidence for a physiological function of HuR, and highlights its involvement in spermatogenesis.

Neuronal promoter of human aromatic L-amino acid decarboxylase gene directs transgene expression to the adult floor plate and aminergic nuclei induced by the isthmus.

In order to analyze the regulatory sequences involved in the neuronal expression of aromatic L-amino acid decarboxylase (AADC), we have generated transgenic mice carrying the LacZ gene under the control of a 3.6-kb human aadc genomic fragment flanking the neuronal alternative first exon. A series of double labeling experiments were performed to compare the pattern of transgene expression to that of specific markers for catecholaminergic and serotonergic neurons. In the adult brain parenchyma, transgene expression was observed in the substantia nigra (SN), the ventral tegmental area (VTA) and the dorsal, medial and pontine raphe nuclei. A large degree of co-expression was observed with tyrosine-hydroxylase (TH) in the SN and VTA, and with serotonin (5-HT) in the dorsal raphe nucleus. Moreover, expression was observed in cells that were both TH- and 5-HT-negative, in particular in the ventral tegmental decussation and the dorsal tip of the VTA. Transgene expression was also observed in the walls of central cavities. Cells positive for both beta-gal and PSA-NCAM were localized in the ventral ependyma of the third and fourth ventricle, and of the central canal of the spinal cord, in what appears to be the adult floor plate. Transgene expressing, PSA-NCAM negative, cells located along the ventral midline of the spinal cord seemed to have migrated out of the ependyma. Our data thus reveal the complexity of aadc gene regulation. The present transgene provides a unique marker for monoaminergic nuclei induced by the isthmus and for the adult floor plate.