The TLS/FUS gene is involved in a recurrent chromosomal translocation in human myxoid liposarcomas. We previously reported that TLS is a potential splicing regulator able to modulate the 5-splice site selection in an E1A pre-mRNA. Using an in vitro selection procedure, we investigated whether TLS exhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this motif abolishes the interaction of TLS with the selected RNAs. We showed that TLS can bind GGUG-containing RNAs with a 250 nM affinity. By UV cross-linking/competition and immunoprecipitation experiments, we demonstrated that TLS recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contributes to the specificity of the TLS⅐RNA interaction, whereas only RRM and RGG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS⅐RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the -tropomyosin pre-mRNA. Moreover, we determined that RNA binding specificities of TLS and high nuclear ribonucleoprotein A1 were different. Hence, our results help define the role of the specific interaction of TLS with RNA during the splicing process of a pre-mRNA. TLS (Translocated in LipoSarcoma)1 or FUS has been first characterized as a rearranged gene in chromosomal translocations specific of human myxoid liposarcoma (1, 2). The resulting fusion protein contains the N-terminal part of TLS fused to a transcription factor of the CAAT/enhancer-binding protein family of proteins: CHOP. In an acute myeloid leukemia, TLS is also involved in a chromosomal breakpoint that juxtaposes the same N-terminal region of TLS to a transcription factor of the ETS proteins family: ERG-1 (3, 4). TLS is highly similar to EWS, a gene implicated in chromosomal translocations that are specific of the tumors of the Ewing family (5, 6). In most of these sarcoma, the N-terminal region of EWS is fused to the DNA binding domain of either ERG-1 or FLI-1, which are two closely related ETS proteins.Both TLS and EWS have in common a similar structural organization. Their C terminus part contains multiple domains that are involved in RNA⅐protein interactions: an RNA recognition motif (RRM) flanked by two regions rich in Arg-Gly-Gly repeats (RGG domains) and a C 2 C 2 zinc finger. In their N terminus domain, they contain a glutamine-, serine-, and tyrosine-rich region that functions as a transcriptional activation domain when fused to a heterologous DNA binding domain (7-9). In oncogenic chimera, the adjunction of this N-terminal region of TLS or EWS to the transcriptional regulators CHOP, FLI-1, or ERG-1 generates proteins with transcriptional activities that differ from those of the wild-type counterpart (8,10). From these data it has been proposed that the oncogenic fusion proteins disturb the expression of genes that are regulated by CHOP, FLI-1, or ERG-1. However, no cellular targe...
Spi-1/PU.1 is an Ets protein deregulated by insertional mutagenesis during the murine Friend erythroleukemia. The overexpression of the normal protein in a proerythroblastic cell prevents its terminal differentiation. In normal hematopoiesis Spi-1/PU.1 is a transcription factor that plays a key role in normal myeloid and B lymphoid differentiation. Moreover, Spi-1/PU.1 binds RNA and interferes in vitro with the splicing process. Here we report that Spi-1 interacts in vivo with TLS (translocated in liposarcoma), a RNA-binding protein involved in human tumor-specific chromosomal translocations. This interaction appears functionally relevant, since TLS is capable of reducing the abilities of Spi-1/ PU.1 to bind DNA and to transactivate the expression of a reporter gene. In addition, we observe that TLS is potentially a splicing factor. It promotes the use of the distal 5 splice site during the E1A pre-mRNA splicing. This effect is counterpoised in vivo by Spi-1. These data suggest that alteration of pre-mRNA alternative splicing by Spi-1 could be involved in the transformation of an erythroblastic cell.The transcription factor Spi-1/PU.1 (1) is an Ets protein that plays a central role in the differentiation of macrophages and B cells during normal hematopoiesis. Indeed, the disruption of the Spi-1/PU.1 gene in mouse (2, 3) induces an early lethality of animals that lack mature macrophages, neutrophils, B and T cells, and also osteoclasts (4). These animals present no alteration in erythrocytic and megacaryocytic lineages. Consistent with this phenotype, Spi-1-responsive elements have been identified in transcriptional promoters and enhancers of many myeloid and B lymphoid genes (for review, see Ref. 5). In murine Friend acute erythroleukemia, the mutation of the spi-1 gene by retroviral insertional mutagenesis induces its transcriptional activation. The erythroleukemic process developed in spi-1 transgenic mice reveals that Spi-1 is involved in blocking the differentiation of the proerythroblast (6). The molecular mechanisms that lead Spi-1 to arrest the erythroid differentiation are not yet understood. The up-regulation of Spi-1 related to the transformation of the proerythroblast suggests that Spi-1 may induce a transcriptional dysregulation of some erythroid genes or/and may be abnormally associated with erythroid partners.Some nuclear proteins interacting with Spi-1 have already been identified such as the transactivator NF-IL6/C/EBP␦ (7), the transcription factor Pip/NF-EM5 (8), the retinoblastoma protein (9), and the basal transcription factor TFIID (9). We reported previously that Spi-1 interacts with the RNA-binding protein p54 nrb (nuclear RNA-binding protein, 54 kDa), binds to RNA by its DNA-binding domain and interferes in vitro with the splicing of a -globin minigene (10). Here, we describe that Spi-1/PU.1 interacts with another RNA-binding protein TLS and that this association impedes the transcriptional functions of Spi-1. In addition, TLS influences, in vivo, the selection of the 5Ј splice site dur...
We have used the human leukemia cell line K562 as a model to study the role of c-myc in di erentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can di erentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid di erentiation markers, without any detectable e ects in the myelomonocytic di erentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid di erentiation, thus reproducing the e ects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/ Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undi erentiated state and to undergo drug-mediated apoptosis.
We present a cytological and biochemical study of the cell death of granule cell precursors in developing rat cerebellum following treatment with the cytotoxic agent methylazoxymethanol (MAM) during the first postnatal week. The density of apoptotic figures per square millimeter progressively increases after 6, 12, 24 and 44 h of treatment, whereas cells immunoreactive for proliferating cell nuclear antigen tend to disappear in the external granular layer (EGL). DNA migration on gel electrophoresis reveals a typical ladder pattern of internucleosomal cleavage following MAM treatment, whereas gel electrophoresis of rRNA shows a conspicuous degradation of both 28S and 18S rRNAs. Ultrastructural analysis has revealed the alterations of structures containing chromatin and ribonucleoprotein (RNP) in dying cells of the EGL. The typical granular beaded configuration of the condensed chromatin changes to a denser, more homogeneous texture suggesting nucleosomal disruption. The reorganization of RNP nuclear domains is reflected by the appearance of dispersed nucleoplasmic RNP particles and the formation of a coiled-body-like structure. However, typical nuclear domains involved in the splicing of RNAs, namely interchromatin granule clusters and typical "coiled bodies", are not found in apoptotic cells. Intranuclear bundles of filaments have also been detected. In the cytoplasm, the presence of dispersed single ribosomes is an initial sign of apoptosis. The massive dispersion and disruption of ribosomes detected after 24 h and 44 h of MAM treatment is reflected by the degradation of both 28S and 18s rRNAs. These results show that MAM treatment provides a useful experimental model for the study of apoptosis in the developing central nervous system. The organization of the cell nucleus in cells undergoing apoptosis clearly reflects a disruption of the nuclear compartments involved in transcription and the processing and transport of RNA and is related to the patterns of DNA and rRNA degradation.
2. 13 On the other hand, Haldar et al 14 described that treatment this report, we found that OA induced apoptosis in three Keywords: Bcl-2; Bcl-XL; apoptosis; okadaic acid; myeloid myeloid leukemia cell lines, which was accompanied by down-regulation of Bcl-2, Bcl-X L and Bax. Furthermore, we also demonstrated that overexpression of either Bcl-X L or BclIntroduction 2 blocked OA-induced apoptosis in K562 cells.Phosphorylation and dephosphorylation of cellular proteins are implicated in many biologically important processes such Materials and methods as cell growth and differentiation. Protein phosphorylation has been recently implicated in pathways that lead to apoptosis. Cell cultureApoptosis is induced in U937 myeloid leukemia cells by cellpermeable analogues of ceramide. 1 One of the candidate K562, KU812 and HL-60 myeloid cell lines were maintained ceramide-regulated targets is ceramide-activated protein phosin RPMI 1640 medium (Seromed; Biochrom KG, Berlin, phatase (CAPP), which belongs to the phosphatase class 2A Germany) supplemented with 10% heat-inactivated fetal calf (PP2A) family of serine/threonine protein phosphatases. 2 Furserum (Flow Laboratories, Irvine, UK), non-essential aminothermore, ceramide-induced apoptosis is inhibited by okadaic acids, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml acid (OA), an inhibitor of serine/threonine protein phosphastreptomycine. Viability and total cell counts were determined tases, being more potent on the PP2A type phosphatases. [2][3][4] at various times by trypan blue exclusion and counting of at Okadaic acid also inhibits glucocorticoid-induced apoptosis least 200 cells from each individual culture. Cells (5-9 × 10 5 in murine T cell hybridomas 5 and apoptosis induced in B cell cells/ml) were cultured in the presence of 15 nM okadaic acid lymphoma and T cell leukemia lines by either heat treatment (Boehringer Mannheim, Mannheim, Germany) for various or ionizing radiation. 6 Apoptosis of lymphoid tumor cells is times. In some experiments, cells were treated with 5 nM accompanied by dephosphorylation of a limited number of calyculin A (Boehringer Mannheim). specific proteins and OA is able to prevent apoptosis and protein dephosphorylation in these cells. 6 On the contrary, this inhibitor of protein phosphatases has been described to Apoptotic cell death analysis induce apoptosis in different mammalian cells, 7 mouse fibroblasts, 8 human breast tumor cells, 9 HeLa cells, 10 and K562, Morphological characteristics of apoptosis were examined HL-60 and U937 myeloid leukemic cells. 11,12 Thus OA has under a light microscope following 48 h of treatment with OA. opposite effects on apoptosis depending on the cell system For the DNA fragmentation analysis, cells were treated as studied.described previously. 17 Briefly, cell pellets were resuspended in lysis buffer containing 0.5% SDS and centrifuged. Supernatants were incubated with 0.5 mg/ml proteinase K,
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