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Abstract Background The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

Abstract In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CL Pro of coronaviruses, so that the luminescent biosensor is turned on upon 3CL Pro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

Human immunodeficiency virus type 1 (HIV-1) unspliced mRNA drives the expression of both Gag and Gag-Pol polyproteins by using both cap- and internal ribosome entry site (IRES)-dependent translation initiation mechanisms. An IRES has been described in the matrix coding region that is involved in the production of shorter isoforms of Gag. However, up to now, this has only been shown with sequences derived from the HIV-1 laboratory strains (NL4.3 and HXB2) and never from clinical HIV-1 isolates. We have isolated ~70 sequences from HIV-1-positive patients that we have sequenced and cloned into an expression vector to monitor their ability to drive translation of Gag p55 and the shorter isoforms both in vitro and ex vivo. The results indicate that (1) the translational efficiency from the AUG-p55 varies significantly among the different isolates; (2) expression initiated at AUG-p40 codon is independent of translation initiation at the AUG-p55 triplet; and (3) all sequences promote expression of shorter Gag isoforms, in particular in Jurkat T cells, in which internal initiation occurs exclusively and directly at the AUG-p40 codon. The composition of the first ~800 nucleotides of the HIV-1 unspliced mRNA modulates the expression initiated both at the AUG-p55 and AUG-p40 codons and may impact viral production and replication. Interestingly, the AUG-p40 codon and its surrounding nucleotide context are conserved amongst clinical isolates and are used as a translation initiation site to produce a shorter Gag isoform.

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity., Competing Interests: The authors have declared that no competing interests exist.

Background: Only a handful of signaling pathways are major actors of development and responsible for both the conservation and the diversification of animal morphologies. To explain this twofold nature, gene duplication and enhancer evolution were predominantly put forth as tinkering mechanisms whereas the evolution of alternative isoforms has been, so far, overlooked. We investigate here the role of gain and loss of isoforms using Edaradd, a gene of the Ecodysplasin pathway, implicated in morphological evolution. A previous study had suggested a scenario of isoform gain and loss with an alternative isoform (A) newly gained in mammals but secondarily lost in mouse lineage., Results: For a comprehensive view of A and B Edaradd isoforms history during mammal evolution, we obtained sequences for both isoforms in representative mammals and performed in vitro translations to support functional predictions. We showed that the ancestral B isoform is well conserved, whereas the mammal-specific A isoform was lost at least 7 times independently in terminal lineages throughout mammal phylogeny. Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level. Finally, only B isoform could rescue a zebrafish edaradd knockdown., Conclusions: These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics. We speculate that during mammal diversification, A isoform regulation may have evolved rapidly, accompanying and possibly supporting the diversity of ectodermal appendages, while B isoform may have ensured essential roles. This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1 h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.

During the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells. This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding protein and stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observed that increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a 'packageable' gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The protein of retroviral origin ENS-1/ERNI plays a major role during neural plate development in chick embryos by controlling the activity of the epigenetic regulator HP1γ, but its function in the earlier developmental stages is still unknown. ENS-1/ERNI promoter activity is down-regulated upon differentiation but the resulting protein expression has never been examined. In this study, we present the results obtained with custom-made antibodies to gain further insights into ENS-1 protein expression in Chicken embryonic stem cells (CES) and during their differentiation. First, we show that ENS-1 controls the activity of HP1γ in CES and we examined the context of its interaction with HP1γ. By combining immunofluorescence and western blot analysis we show that ENS-1 is localized in the cytoplasm and in the nucleus, in agreement with its role on gene's promoter activity. During differentiation, ENS-1 decreases in the cytoplasm but not in the nucleus. More precisely, three distinct forms of the ENS-1 protein co-exist in the nucleus and are differently regulated during differentiation, revealing a new level of control of the protein ENS-1. In silico analysis of the Ens-1 gene copies and the sequence of their corresponding proteins indicate that this pattern is compatible with at least three potential regulation mechanisms, each accounting only partially. The results obtained with the anti-ENS-1 antibodies presented here reveal that the regulation of ENS-1 expression in CES is more complex than expected, providing new tracks to explore the integration of ENS-1 in CES cells regulatory networks.

Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m(7)GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5' of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5' free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation.

Expression of the two isoforms p55 and p40 of HIV-1 Gag proteins relies on distinct translation initiation mechanisms, a cap-dependent initiation and two internal ribosome entry sites (IRESs). The regulation of these processes is complex and remains poorly understood. This study was focused on the influence of the 5'-UTR and on the requirement for the eukaryotic initiation factor (eIF)4F complex components. By using an in vitro system, we showed substantial involvement of the 5'-UTR in the control of p55 expression. This highly structured 5'-UTR requires the eIF4F complex, especially RNA helicase eIF4A, which mediates initiation at the authentic AUG codon. In addition, the 5'-UTR regulates expression in an RNA concentration-dependent manner, with a high concentration of RNA triggering specific reduction of full-length Gag p55 production. HIV-1 genomic RNA also has the ability to use a strong IRES element located in the gag coding region. We show that this mechanism is particularly efficient, and that activity of this IRES is only poorly dependent on RNA helicase eIF4A when the viral 5'-UTR is removed. HIV-1 genomic mRNA exhibits in vitro translational features that allow the expression of Gag p55 protein by different mechanisms that involve different requirements for eIF4E, eIF4G, and eIF4A. This suggests that HIV-1 could adapt to its mode of translation according to the availability of the initiation factors in the infected cell., (© 2012 The Authors Journal compilation © 2012 FEBS.)

The 5'-untranslated region (5'-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5'-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5'-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

BRCA1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair, cell cycle control, and apoptosis. We identified poly(A)-binding protein 1 (PABP) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by in vitro assays and coimmunoprecipitation in mammalian cells. Endogenous interaction between BRCA1 and PABP was also observed. This interaction was abolished by BRCA1 cancer-associated mutations, suggesting that it may be physiologically relevant. Deletion mapping demonstrated that the RNA recognition motifs 1-4 region of PABP is required to mediate the interaction with BRCA1. To understand the biological function of the BRCA1-PABP complex, we sought to determine whether BRCA1 is a modulator of translation. We showed here that inhibition of endogenous BRCA1 using a small interfering RNA-based approach decreased protein synthesis. Conversely, overexpression of BRCA1 activated translation. Using a RNA transfection approach, we clearly showed that BRCA1 modulates translation, independently of any transcriptional activity. The data presented here suggest that BRCA1 modulates protein synthesis via its interaction with PABP, providing a novel mechanism by which BRCA1 may exert its tumor suppressor function.

Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5' cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5' untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5' untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.

In the following three sections, we will briefly review the seminal roles of the HIV-1 nucleocapsid protein p7 (NCp7) in the fate of the HIV-1 full length RNA from genomic RNA in a dimeric form to the proviral DNA. Emphasis will be given to the mechanisms of NC-directed assistance to the genomic RNA and reverse transcriptase (RT) in the course of proviral DNA synthesis and to DNA integrity at the end of the polymerization process, and to the NC-assisted repair and recombination reactions fueling the viability and variability of the virus., (Copyright 2004 Elsevier B.V.)

The eukaryotic translation initiation factor eIF4GI binds several proteins and acts as a scaffold to promote preinitiation complex formation on the mRNA molecule (48S). Following mRNA attachment this complex scans along the messenger in a 5' to 3' direction until it locates and recognizes the initiation start codon. By using a combination of retroviral and picornaviral proteases (HIV-2 and L respectively) in the reticulocyte lysate system, we have characterized a 40 amino acid (aa) region of eIF4GI (aa 642-681) that exhibits general RNA-binding properties. Removal of this domain by proteolytic processing followed by translational assays showed virtually no inhibition of internal ribosome entry on the encephalomyocarditis virus, but resulted in drastic impairment of ribosome scanning as demonstrated by studying poliovirus and foot-and-mouth disease virus translation. Based on these findings, we propose that this 40 aa motif of eIF4GI is critical for ribosome scanning.

Objective: To assess the prevalence of white-coat normortension, white-coat hypertension, and white-coat effect., Methods: We assessed 670 medical records of patients from the League of Hypertension of the Hospital das Clínicas of the Medical School of the University of S o Paulo. White-coat hypertension (blood pressure at the medical office: mean of 3 measurements with the oscillometric device > or = 140 or > or = 90 mmHg, or both, and ambulatory blood pressure monitoring mean during wakefulness < 135/85) and white-coat normotension (office blood pressure < 140/90 and blood pressure during wakefulness on ambulatory blood pressure monitoring > or = 135/85) were analyzed in 183 patients taking no medication. The white-coat effect (difference between office and ambulatory blood pressure > 20 mmHg for systolic and 10 mmHg for diastolic) was analyzed in 487 patients on treatment, 374 of whom underwent multivariate analysis to identify the variables that better explain the white-coat effect., Results: Prevalence of white-coat normotension was 12%, prevalence of white-coat hypertension was 20%, and prevalence of the white-coat effect was 27%. A significant correlation (p<0.05) was observed between white-coat hypertension and familial history of hypertension, and between the white-coat effect and sex, severity of the office diastolic blood pressure, and thickness of left ventricular posterior wall., Conclusion: White-coat hypertension, white-coat normotension, and white-coat effect should be considered in the diagnosis of hypertension.

In eukaryotic cells, protein synthesis is regulated by a set of initiation factors (eIF) that are required for recruiting the 40 S ribosomal subunit onto the mRNA molecule. Among these proteins, eIF4GI, which is targeted by picornaviral proteases, makes a bridge between the mRNA cap structure (via eIF4E) and the 40 S ribosome (via eIF3). Recently, internal ribosome entry segment (IRES) elements have been characterized in the genomic RNA of both simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), suggesting that viral expression of these two viruses can be regulated at the translational level. Thus, by analogy with members of the picornavirus family, we have investigated the action of the HIV-1 protease on initiation factors eIF4GI and eIF4GII using cell extracts and the rabbit reticulocyte lysate system. Our results show that eIF4GI, but not eIF4GII, is substrate for HIV-1 protease and this effect can be prevented by a HIV-1 protease inhibitor, palinavir. However, in contrast to picornaviral proteases, the cleavage of eIF4GI by HIV-1 protease occurs at multiple sites and impairs translation of both cap-dependent and IRES-containing RNAs, except for the HCV IRES, which does not require eIF4GI or eIF4GII for activity., (Copyright 2002 Elsevier Science Ltd.)

Using the HeLa cell model along with an in vitro splicing system, we have previously shown that hnRNP M and 2H9 are involved in the pre-mRNA splicing process and most interestingly also in heat shock-induced transient splicing arrest by transiently leaving the hnRNP complexes. Due to this unique regulatory function in a mechanism that turns splicing on and off, these two hnRNPs appear as important proteins for controlling gene expression. Here we investigated by in situ hybridization and immunohistochemical staining techniques the expression level of specific mRNA and protein during mouse embryonic development. HnRNP M and 2H9 are found to be expressed at all examined stages (6.5-18.5 days post-coïtum), in a differential manner, and at various levels depending on tissues, cell types and also embryonic stages; fairly high levels of both hnRNPs are always observed in the central nervous system. Furthermore, levels of colocalizing protein and transcript are not always present in the same proportion, thus suggesting a post-transcriptional regulation of hnRNP M and 2H9 gene expression. The complex spatiotemporal variations we observed might well anticipate a role for these two hnRNPs also in modulating splicing, thereby influencing gene expression and further many physiological processes.

The three murine retinoic acid receptor (RAR) genes each contain two distinct promoters which give rise to protein isoforms differing in their N-terminal regions. This study used in situ hybridization to describe the expression patterns of RARalpha1, RARalpha2, RARbeta1/3, RARbeta2/4, RARgamma1 and RARgamma2 isoform transcripts during mouse embryogenesis. RARalpha1 transcripts are widely distributed, with the exception of the central nervous system. Highest expression is found in developing muscle, pituitary gland and various epithelia. On the other hand, RARalpha2 is essentially expressed along the spinal cord up to the hindbrain 7th rhombomere and in the 4th rhombomere, pons and developing basal ganglia (corpus striatum and pallidum). RARbeta2/4 transcripts account for most of the previously described RARbeta expression features being expressed specifically, or more prominently than RARbeta1/3, in foregut endoderm and its derivatives, olfactory and periocular mesenchyme, urogenital region, proximal limb bud mesenchyme and later within interdigital regions. RARbeta1/3 is more prominently expressed in the developing heart outflow tract mesenchyme, intervertebral disks, midgut loop mesenchyme and umbilical vessel walls. RARbeta1/3 and RARbeta2/4 are coexpressed in the developing corpus striatum. They exhibit, however, distinct dorsoventral distributions along the spinal cord and caudal hindbrain. RARgamma2 is the RARgamma isoform expressed at high levels in the caudal neural groove at embryonic day 8.5. At later stages, both RARgamma isoforms are essentially coexpressed, although the progressive restriction of RARgamma1 transcripts to craniofacial or limb precartilaginous condensations appears to precede that of RARgamma2.

To investigate the relative contribution of retinoic acid receptor (RAR)beta isoforms in conotruncal septation, RAR beta 1 and beta 3 were inactivated in the mouse. Mice lacking RAR beta 1 and beta 3 appear normal. Disruption of these isoforms in RAR alpha or RAR gamma null genetic backgrounds results in a high postpartum lethality. However, except for ocular defects found in RAR beta 1-3/RAR gamma compound mutants, the double null mutants display only abnormalities seen in single null mutants. This probably reflects a functional redundancy with other RARs, most notably with RAR beta 2 which is five- to sixfold more abundant than RAR beta 1 and beta 3 and whose domain of expression is largely overlapping. The conotruncal ridges form normally in retinoid X receptor (RXR)alpha/RAR beta compound mutants but fail to fuse, apparently as a result of excessive apoptosis of mesenchymal cells. Additionally, many cardiomyocytes in the conotruncal wall of these mutants appear necrotic. Although RAR beta 1 and beta 3 are expressed specifically in the conotruncal ridges, failure of fusion of these structures is not more frequent in RXR alpha/RAR beta 1-3 double null mutants than in RXR alpha single null mutants. Similarly, the disruption of the sole RAR beta 2 isoform in a RXR alpha null genetic background does not result in an increase of the frequency of conotruncal septum agenesis. However, this agenesis is fully penetrant in RXR alpha/RAR beta +/- mutants, which reflects distinct role of RXR alpha:RAR beta 1 (and beta 3) and RXR alpha:RAR beta 2 heterodimers in promoting the survival of conotruncal mesenchymal cells. Unexpectedly, we discovered that, in wild-type embryos, the conotruncal mesenchyme is a major site of morphogenetic cell death and that conotruncal myocytes are occasionally necrotic. Thus, excessive cell death in the conotruncus is a potential cause of ventricular septal defects in humans.

Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in P19 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.

Retinoids are known to prevent mammary carcinogenesis in rodents and inhibit the growth of human breast cancer cells in vitro. Previously we demonstrated that retinoid inhibition of proliferation of human breast cancer cell lines is largely mediated by retinoic acid receptor (RAR)-alpha. In this study we describe for the first time the histological distribution of RAR-alpha in 33 breast lesion specimens as determined by immunostaining with RAR-alpha antibody. Nuclear staining was observed in tumor tissue and normal portions of the breast samples. Connective tissue exhibited relative uniform staining, whereas a wide range of RAR-alpha expression was found in the epithelial tumor cells. RAR-alpha protein was expressed at significantly higher levels in tumors with greater proliferative activity as determined by immunostaining with Ki-67 antibody. This suggests that RAR-alpha expression may be altered with tumor progression. Although a positive correlation between RAR-alpha mRNA levels and estrogen receptor status of breast tumors has previously been documented, we did not find such a relationship at the protein level. As RAR-alpha plays a major role in retinoid-mediated growth inhibition of human breast cancer cell in vitro, our findings suggest that patients with highly proliferating tumors could be responsive to retinoid independently of their responsiveness to (anti)-estrogens.

Using a differential subtractive hybridization cloning procedure we have recently identified the AP-2.2 gene as a novel early retinoic acid-induced gene in murine P19 embryonal carcinoma cells. We have also shown that the AP-2.2 protein, which is highly related to the AP-2 transcription factor, can activate transcription when bound to an AP-2 consensus binding site [Oulad-Abdelghani et al. (1995) Mol. Cell. Biol., submitted]. We report here the in situ hybridization pattern of expression of AP-2.2 transcripts during mouse embryogenesis. At 7.5 days post-coitum, AP-2.2 transcripts were detected in the boundary region between neural plate and surface ectoderm, as well as in extra-embryonic tissues. By 8.0-8.5 gestational days, AP-2.2 transcripts appeared to be expressed in premigratory and migrating neural crest cells. Over the following days, the AP-2.2 gene displayed region-restricted expression in the facial mesenchyme, especially around the embryonic mouth cavity and the nasal cavities, as well as in the surface ectoderm, nasal and oral epithelia. AP-2.2 RNA was also specifically expressed in the presumptive cortical region of the forebrain vesicles. AP-2.2 transcripts were restricted to the distal mitotic area (the 'progress zone') of the limb buds and of the genital bud. AP-2.2 expression also appeared to be specific for primordial germ cells in the genital ridges. Thus, the AP-2.2 gene is expressed in several embryonic areas whose development can be affected by retinoids, such as the forebrain, face and limb buds.

The cellular retinoic acid binding proteins I and II (CRABPI and CRABPII) bind retinoic acid with high affinity, exhibit distinct patterns of expression during embryonic development, and are thought to play important roles in the RA signaling pathway. We have generated a targeted mutation of the CRABPI gene using the "hit-and-run" strategy and shown that it prevents the production of a functional CRABPI protein. Homozygous mutant mice were normal, indicating that CRABPI does not play a crucial role in the RA signaling pathway.

The developmental expression of retinoic acid (RA) nuclear receptors RAR(alpha, beta, gamma) and RXR(alpha, beta, gamma) was analysed during mouse odontogenesis by in situ hybridization on frozen sections and compared with the expression patterns of the cellular retinoic acid binding proteins CRABPI and II. The transcripts distribution of each RAR and RXR was basically similar in developing molars and incisors. RAR alpha and RXR alpha were preferentially expressed in dental epithelia, whereas RAR gamma and RXR gamma were transcribed in the dental mesenchyme. RAR beta, RAR gamma and RXR beta displayed both epithelial and mesenchymal expression. RAR beta expression was initiated during bell stage. RXR gamma transcripts were observed only at day 19.5 post coitum in the mitogenic mesenchyme facing the epithelial loops. Odontoblasts expressed RAR beta and RAR gamma, RXR alpha and RXR beta. Preameloblasts expressed RXR alpha and RXR beta and ameloblasts RXR gamma, RXR alpha and RXR beta. RAR alpha transcription in the incisor preameloblasts and ameloblasts was not observed in the first molar. The coexpression between RARs and RXRs might be important to form RAR/RXR heterodimers which are necessary to activate the transcriptions of target genes. CRABPI and CRABPII demonstrated graded variation of expression during odontogenesis in the mesenchyme and in the inner dental epithelium respectively. The pattern of CRABPI transcripts overlapped at least partially with expressions of all the studied nuclear receptors whereas CRABPII epithelial expression was superimposed with the transcription of RAR alpha, RXR alpha and RXR beta. These cytoplasmic proteins might participate in the storage and/or metabolism of RA and then distribute RA to colocalized nuclear receptors.(ABSTRACT TRUNCATED AT 250 WORDS)