Wnt/ß-catenin pathway and cell adhesion deregulation in CSDE1-related intellectual disability and autism spectrum disorders.

Among the genetic factors playing a key role in the etiology of intellectual disabilities (IDs) and autism spectrum disorders (ASDs), several encode RNA-binding proteins (RBPs). In this study, we deciphered the molecular and cellular bases of ID-ASD in a patient followed from birth to the age of 21, in whom we identified a de novo CSDE1 (Cold Shock Domain-containing E1) nonsense variation. CSDE1 encodes an RBP that regulates multiple cellular pathways by monitoring the translation and abundance of target transcripts. Analyses performed on the patient's primary fibroblasts showed that the identified CSDE1 variation leads to haploinsufficiency. We identified through RNA-seq assays the Wnt/ß-catenin signaling and cellular adhesion as two major deregulated pathways. These results were further confirmed by functional studies involving Wnt-specific luciferase and substrate adhesion assays. Additional data support a disease model involving APC Down-Regulated-1 (APCDD1) and cadherin-2 (CDH2), two components of the Wnt/ß-catenin pathway, CDH2 being also pivotal for cellular adhesion. Our study, which relies on both the deep phenotyping and long-term follow-up of a patient with CSDE1 haploinsufficiency and on ex vivo studies, sheds new light on the CSDE1-dependent deregulated pathways in ID-ASD.

Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells.

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation. However, after ionizing radiation (IR), clonogenic survival of unr(-/-) ES cells was approximately 3-fold enhanced as compared to unr(+/+) cells. We further determined that IR-induced apoptosis was decreased in unr(-/-) ES cells, and that reintroduction of the unr gene in unr(-/-) cells restored normal IR-induced apoptosis. Three pro-apoptotic genes, p53, caspase-3 and Gadd45gamma, were downregulated in unr(-/-) ES cells, indicating that Unr, as other cytoplasmic RNA-binding proteins, regulates a complex genetic program, promoting cell death after IR. In contrast, in the human hepatoma cell line HuH7, Unr knockdown using unr-specific small interfering RNAs induced apoptosis, both in untreated and gamma-irradiated cells. Thus, our results establish that Unr acts as a positive or negative regulator of cell death, depending on the cell type. Manipulating the level of Unr may constitute a specific approach to sensitize cancer cells to anticancer treatments.

A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex.

mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblasts with human topoisomerase IIalpha cDNA: selective restoration of the sensitivity to DNA religation inhibitors.

In the Chinese hamster lung cell line DC-3F/9-OH-E, selected for resistance to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase IIalpha is 4-5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIbeta is undetectable. Cloning and sequencing of topoisomerase IIalpha cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr). To analyze the contribution of these topoisomerase IIalpha alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase IIalpha cDNA. In one transfected clone, the amount of topoisomerase IIalpha isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase IIalpha, are then a unique mammalian cell line to analyze the physiological and pharmacological properties of this enzyme. However, the restoration of a nearly normal topoisomerase IIalpha activity in the DC-3F/9-OH-E cells did not have the same effect on their sensitivity to different enzyme inhibitors; a 75% reversion of the resistance, associated with a 2-3-fold increased stabilization of the cleavable complex, was observed with both etoposide and m-AMSA, two drugs that inhibit the DNA religation step in the enzyme catalytic cycle; in contrast, the transfected cells remained fully resistant to ellipticine derivatives that did not induce the stabilization of the cleavable complex. We hypothesized that a trans-acting factor, inhibiting the induction of cleavable complex formation by drugs that are not religation inhibitors, might be present in the resistant cells. However, such a factor was not detected in in vitro experiments, and other hypotheses are discussed.

RNA binding specificity of Unr, a protein with five cold shock domains.

The human unr gene encodes an 85 kDa protein which contains five cold shock domains (CSD). The capacity of Unr to interact in vitro with RNA and its intracellular localization suggest that Unr could be involved in some aspect of cytoplasmic mRNA metabolism. As a step towards identification of Unr mRNA targets, we investigated the RNA-binding specificity of Unr by an in vitro selection approach (SELEX). Purine-rich sequences were selected by Unr, leading to the identification of two related consensus sequences characterized by a conserved core motif AAGUA/G or AACG downstream of a purine stretch. These consensus sequences are 11-14 nt long and appear unstructured. RNAs containing a consensus sequence were bound specifically by Unr with an apparent dissociation constant of 1 x 10(-8) M and both elements, the 5' purine stretch and the core motif, were shown to contribute to the high affinity. When the N-terminal and C-terminal CSD were analyzed individually, they exhibited a lower affinity than Unr for winner sequences (5- and 100-fold, respectively) but with similar binding specificity. Two combinations of CSDs, CSD1-2-3 and CSD1*2-3-4-5 were sufficient to achieve the high affinity of Unr, indicating some redundancy between the CSDs of Unr for RNA recognition. The SELEX-generated consensus motifs for Unr differ from the AACAUC motif selected by the Xenopus Y-box factor FRGY2, indicating that a diversity of RNA sequences could be recognized by CSD-containing proteins.

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity.

mRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3-4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3-4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3-4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3-4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.

Transcription of unr (upstream of N-ras) down-modulates N-ras expression in vivo.

The ras proteins (Harvey, Kirsten and N-ras) are key regulators of signal transduction and a perturbation of their GDP/GTP cycle is frequently observed in tumors. In mammals, N-ras constitutes with unr (upstream of N-ras) a tightly linked tandem of ubiquitously expressed genes. Although unr and N-ras appear to be involved in distinct functions, this unusual genetic organization could be important for the regulation of N-ras expression. Specifically, transcription of unr could negatively regulate that of N-ras by transcriptional interference. To investigate this possibility, we have deleted the unr promoter by homologous recombination in murine embryonic stem cells. Analysis of tissues of heterozygous mice revealed an increase in N-ras mRNA accumulation ranging between 20 and 65%, in agreement with the suppression of a transcriptional interference.

Nucleic acid binding and intracellular localization of unr, a protein with five cold shock domains.

The unr gene was identified as a transcription unit located immediately upstream of N-ras in the genome of several mammalian species. While this genetic organization could be important for the transcriptional regulation of unr and N-ras, the function of the protein product of unr is unknown. unr is ubiquitously expressed and codes for an 85 kDa protein which is not closely related to previously characterized proteins. Nevertheless, a search for protein motifs has indicated the presence of five 'cold shock domains' within unr, a motif present in procaryotic cold shock proteins and in the vertebrate Y box factors. As these proteins have been reported to interact with nucleic acids, we investigated whether unr could bind to some classes of nucleic acids. We report here that unr has a high affinity for single-stranded DNA or RNA and a low affinity for double-stranded nucleic acids. Its low affinity for double-stranded DNA clearly distinguishes unr from the Y box factors. The binding of unr to RNA does not appear to depend upon extended sequence motifs but requires some level of sequence complexity as unr has only a low affinity for most simple polymers including polyA stretches. unr is also characterized by its low affinity for double-stranded and structured RNAs. We further determined that unr is mostly localized in the cytoplasm, and is in part associated with the endoplasmic reticulum. These studies indicate that unr is a novel single-stranded nucleic acid binding protein which is likely to be associated with cytoplasmic mRNA in vivo.

Genomic cloning of human thioredoxin-encoding gene: mapping of the transcription start point and analysis of the promoter.

Thioredoxin (TR) is a small ubiquitous dithiol-reductase enzyme first identified in bacteria and plants. In recent years, this protein has been recognized as playing an important role in the growth control of eukaryotic cells, especially in lymphocytes. It was first cloned from a human Epstein-Barr virus-transformed lymphoblastoid B-cell line by our group in 1988 [Wollman et al., J. Biol. Chem. 263 (1988) 15506-15512] and localized on chromosome 3 p11-p12 by in situ hybridization [Lafage-Pochitaloff-Huvalé et al., FEBS Lett. 255 (1989) 89-91]. The present work was performed to study the genomic organization of the human thioredoxin (hTR)-encoding gene (hTR). The screening of a human genomic library in lambda EMBL4 phage led to the identification of two genomic clones which encompassed the entire gene, including the promoter region. The coding region of hTR spans over 13 kb and is organized into five exons separated by four introns which were 60% sequenced. We determined the transcription start point (tsp) by primer extension. This tsp located, in lymphocytes, 22-bp downstream from a TATA box (TATAA) defines a 5' untranslated region of 74 bp. We analyzed 2149-bp upstream from the promoter for sequence motifs which could bind regulatory proteins. This promoter contains many possible regulatory elements compatible with both a basal constitutive expression and a regulated inducible transcription, especially by cytokines such as interleukin-6 and interferons. Finally, Southern hybridization of genomic DNAs from several donors detected only one active gene encoding hTR.

Exon skipping in the expression of the gene immediately upstream of N-ras (unr/NRU).

The unr (or NRU) gene was identified during investigations of the structure of the N-ras locus in the genome of mammals. A striking feature of the unr/N-ras gene tandem is the short intergenic distance of 150 nucleotides which suggests the possibility of transcriptional interactions between the two genes. At present, the function of the unr gene is unknown, but the predicted translation product shows a distant relationship to a class of DNA binding proteins. Comparison of the two published cDNA sequences, from a human lymphocytic and a rat testis cDNA library, reveals a difference of 31 amino acids in the size of the predicted proteins. We show that this is due to the skipping of exon 5 within the human NRU gene and that a similar phenomenon occurs in the rat unr gene. Exon skipping takes place in all the cells and tissues we have analyzed and generates the predominant form of message, except in the brain where both classes are about as abundant. This exon skipping is independent of other aspects of unr expression such as the choice of the polyadenylation site.

Organization of the unr/N-ras locus: characterization of the promoter region of the human unr gene.

Investigations of the structure and expression of the N-ras gene in mammals has led to the identification of another gene designated unr, which is located immediately upstream of N-ras. These two genes are transcribed in the same orientation and the intergenic distance is of the order of 150 nucleotides. This genetic organization has been observed in the genome of guinea pig, rat, mouse and man with a very high level of sequence conservation in the intergenic region. This unusual gene clustering suggests that the transcriptional regulation of this locus could involve common regulatory sequences as well as transcriptional interference between the two genes. In this study, we have isolated and characterized the human unr promoter. A cluster of transcription initiation sites was mapped by primer extension and RNase protection and shown to be located in a CpG island devoid of TATA and CAAT boxes. Functional organization of the promoter was investigated by measuring the ability of a set of 5' deletions within a1 kb promoter region to drive the expression of the luciferase gene. These studies indicated a very strong promoter activity in NIH 3T3 cells and the presence of positive and negative regulatory domains. Nevertheless, a 90 bp fragment showed the same level of promoter activity as the 1 kb fragment. We also showed that ras genes can transactivate the unr promoter activity and that the 90 bp fragment responded to this transactivation.

Glucocorticoids regulate expression of the v-mycOK10 oncogene in a murine retroviral vector with chimeric MoMuLV-MMTV LTRs.

A murine retrovirus which expresses the v-mycOK10 oncogene under the control of the dexamethasone-regulatable mouse mammary tumor virus (MMTV) promoter has been constructed. In this vector, denoted pMImyc, the Moloney Murine leukemia virus (MoMuLV) sequences required for virus replication, integration and packaging were kept, while all the elements for transcription regulation were derived from the MMTV long terminal repeat (LTR). After transfection of NIH 3T3 fibroblasts with this construct, a cell line was isolated in which the level of v-myc RNAs were increased 60 fold by dexamethasone. Kinetic studies showed that this induction can be maintained for up to 12 hours of hormone treatment. After infection with MoMuLV as a helper virus, and in the presence of dexamethasone, the production of pMImyc RNA, estimated by slot blot analysis, was equivalent to about 10(3) viral particles/ml.

Transfer of immortality by transfection of genomic DNA from SV40 established cell lines into rat embryo fibroblasts.

The cellular immortalization activity of cloned genes can be identified either in a colony-forming assa of transfected primary rat embryo fibroblasts or in a co-operation assay together with ras. However the demonstration of immortalization activities carried by cellular genes has not been reported. Here we establish that SV40 early genes integrated in genomic DNAs can be stably transferred into rat embryo fibroblasts and selected via their immortalization activity. Attempts to extend this assay to the identification of dominant genes putatively involved in the immortality of several other immortal post-crisis or tumor cells have been unsuccessful suggesting that the immortal phenotype can be brought about through different pathways.

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.

Yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide. III. Possible invovlement in the induction of "petite" mutation by phenanthridinium derivatives.

We previously described a yeast-mitochondrial deoxyribonuclease (EtdBr DNAase), whose activity is stimulated by ethidium bromide. In this paper, we have compared the ability of a series of phenanthridinium derivatives to activate the EtdBr DNAase "in vitro" and their efficiency in inducing "petite" mutants in the yeast S. cerevisiae. Kinetics studies, in the absence or the presence of SDS, were first carried out to compare the penetration rates of the various compounds. Dose--response curves were then established to quantify their mutagenic efficiencies. From these data, a linear correlation was established between the level of EtdBr DNAase activation produced by a drug and its mutagenic efficiency, thus demonstrating that the two processes display similar drug-structural requirements. These results suggest that the EtdBr DNAase might be involved in the induction of petite mutations by these derivatives.

Yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide. 2. Mechanism of enzyme activation.

An endonuclease (EtdBr DNase), which is more active in the presence of EtdBr, has been purified from yeast mitochondrial membrane (Jacquemin-Sablon, H., et al. (1979) Biochemistry 18 (preceding paper in this issue)). This paper deals with the analysis of the mechanism of this activation. Determination of the enzyme activity in the presence of intercalating and nonintercalating agents showed that the enzyme does not recognize the DNA structure modifiction provoked by drug intercalation. Studies carried out with a series of phenanthridinium derivatives led to the following model. The EtdBr DNase activation would result from the formation of a ternary complex, DNA--drug--Triton X-100. The activation capacity of a drug depends on its ability to bind simultaneoulsy to the DNA (not necessarily by intercalation) and the detergent. When this complex is formed, the DNA molecule is surrounded with Triton X-100 molecules which constitute an hydrophobic environment and make the substrate more prone to interaction with the enzyme. The implications of this model are discussed.