38 research outputs found

    Compositional Genome Contexts Affect Gene Expression Control in Sea Urchin Embryo

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    Gene expression is widely perceived as exclusively controlled by the information contained in cis-regulatory regions. These are built in a modular way, each module being a cluster of binding sites for the transcription factors that control the level, the location and the time at which gene transcription takes place. On the other hand, results from our laboratory have shown that gene expression is affected by the compositional properties (GC levels) of the isochores in which genes are embedded, i.e. the genome context. To clarify how compositional genomic properties affect the way cis-regulatory information is utilized, we have changed the genome context of a GFP-reporter gene containing the complete cis-regulatory region of the gene spdeadringer (spdri), expressed during sea urchin embryogenesis. We have observed that GC levels higher or lower than those found in the natural genome context can alter the reporter expression pattern. We explain this as the result of an interference with the functionality of specific modules in the gene's cis-regulatory region. From these observations we derive the notion that the compositional properties of the genome context can affect cis-regulatory control of gene expression. Therefore although the way a gene works depends on the information contained in its cis-regulatory region, availability of such information depends on the compositional properties of the genomic context

    Ubiquitin-ligase AIP4 controls differential ubiquitination and stability of isoforms of the scaffold protein ITSN1.

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    At present, the role of ubiquitination of cargoes internalized from the plasma membrane is better understood than the consequences of ubiquitination of proteins comprising the endocytic machinery. Here, we show that the E3 ubiquitin ligase AIP4/ITCH contributes to the differential ubiquitination of isoforms of the endocytic scaffold protein intersectin1 (ITSN1). The major isoform ITSN1-s is monoubiquitinated, whereas the minor one, ITSN1-22a undergoes a combination of mono- and oligoubiquitination. The monoubiquitination is required for ITSN1-s stability, whereas the oligoubiquitination of ITSN1-22a causes its proteasomal degradation. This explains the observed low abundance of the minor isoform in cells. Thus, different modes of ubiquitination regulated by AIP4 have opposite effects on ITSN1 isoform stability

    ITSN1 regulates SAM68 solubility through SH3 domain interactions with SAM68 proline-rich motifs

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    International audienceSAM68 is an mRNA-binding protein involved in mRNA processing in the nucleus that forms membraneless compartmentscalled SAM68 Nuclear Bodies (SNBs). We found that intersectin 1 (ITSN1), a multidomain scaffold protein harboringfive soluble SH3 domains, interacts with SAM68 proline-rich motifs (PRMs) surrounded by self-adhesive low complexitydomains. While SAM68 is poorly soluble in vitro, the interaction of ITSN1 SH3 domains and mRNA with SAM68 enhancesits solubility. In HeLa cells, the interaction between the first ITSN1 SH3 domain (SH3A) and P0, the N-terminal PRM ofSAM68, induces the dissociation of SNBs. In addition, we reveal the ability of another SH3 domain (SH3D) of ITSN1 tobind to mRNAs. ITSN1 and mRNA may thus act in concert to promote SAM68 solubilization, consistent with the absenceof mRNA in SNBs in cells. Together, these results support the notion of a specific chaperoning of PRM-rich SAM68 withinnuclear ribonucleoprotein complexes by ITSN1 that may regulate the processing of a fraction of nuclear mRNAs, notablySAM68-controlled splicing events related to higher neuronal functions or cancer progression. This observation may alsoserve as a putative model of the interaction between other PRM-rich RBPs and signaling proteins harboring SH3 domains

    Identification and functional analysis of an alternative promoter of human intersectin 1 gene

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    Aim. Intersectin 1 (ITSN1) gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional way to create diversity and flexibility in the regulation of gene expression. The aim of this study was to identify possible alternative promoters of ITSN1 gene. Methods. In silico prediction, 5' RACE, RT-PCR and reporter gene expression assay were used for identification and functional characterization of alternative promoter region. Results. We detected an alternative promoter of human ITSN1 gene which is located in intron 5 and generates 5' truncated transcripts containing in-frame ATG codon with strong Kozak sequence and could encode an N-terminally truncated isoforms lacking first EH domain. The region located 246–190 bp upstream of exon 6 is required for alternative promoter activity. ITSN1 transcripts generated from an alternative promoter were detected in human kidney, liver, lung and brain tissues. However, the level of their expression was significantly lower than that of major ITSN1 isoforms. Conclusion. The results obtained suggest that alternative promoter region located in intron 5 of ITSN1 gene functions as a weak promoter. Further experiments are required to clarify the role of 5' truncated ITSN1 transcripts

    RNA-dependent nuclear matrix contains a 33k globin full domain transcript as well as prosomes but no 26S proteasomes

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    Previously, we have shown that in murine myoblasts prosomes are constituents of the nuclear matrix; a major part of the latter was found to be RNase sensitive. Here, we further define the RNA-dependent matrix in avian erythroblastosis virus (AEV) transformed erythroid cells in relation to its structure, presence of specific RNA, prosomes and/ or proteasomes. These cells transcribe but do not express globin genes prior to induction. Electron micrographs show little difference in matrices treated with DNase alone or with both, DNase and RNase. In situ hybridization with alpha globin riboprobes shows that this matrix includes globin transcripts. Of particular interest is that, apparently, a nearly 35 kb long globin full domain transcript (FDT), including genes, intergenic regions and a large upstream domain is a part of the RNAdependent nuclear matrix. The 23K-type of prosomes, previously shown to be co-localized with globin transcripts in the nuclear RNA processing centers, were found all over the nuclear matrix. Other types of prosomes show different distributions in the intact cell but similar distribution patterns on the matrix. Globin transcripts and at least 80% of prosomes disappear from matrices upon RNase treatment. Interestingly, the 19S proteasome modulator complex is insensitive to RNase treatment. Only 20S prosomes but not 26S proteasomes are thus part of the RNA-dependent nuclear matrix. We suggest that giant pre-mRNA and FDTs in processing, aligning prosomes and other RNA-binding proteins are involved in the organization of the dynamic nuclear matrix. It is proposed that the putative function of RNA within the nuclear matrix and, thus, the nuclear dynamic architecture, might explain the giant size and complex organization of primary transcripts and their introns
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