ABSTRACT
[This corrects the article DOI: 10.1371/journal.ppat.1005860.].
ABSTRACT
Because a previous study by conventional cytogenetics had revealed a nullisomy 17 in the breast cancer cell line EFM-19, we analysed that cell line by SKY-FISH and by FISH using different probes derived from chromosome 17. A bicolor FISH using a HER2-specific probe and a chromosome 17 centromeric probe showed five HER2 and six centromeric signals all appearing on different chromosomes A further bicolor FISH using a chromosome 17-specific painting probe and a HER2-specific probe revealed that the HER2 signals were always localized within chromosome 17 segments constituting part of structurally altered chromosomes as deduced from their G-banding. Further FISH analyses using single-locus probes of chromosome 17, i.e., for MDS, p53, SMS and RARA, showed that all five chromosome 17 painting segments contained material from the long arm but only two painting segments had additional material from the short arm. A SKY-FISH confirmed the results of the chromosome 17 painting by FISH, except for one structurally altered chromosome showing additional chromosome 17 material detected by the SKY experiment. These results allow us to conclude that, in this cell line, polysomy 17 has preceeded the fragmentation of chromosome 17 leading to amplification of small parts of that chromosome as well as to extended losses. As to a general mechanism, polysomy 17 and a fragility of this, chromosome in breast cancer cells may not only account for part of the cases with HER2 amplification but, at the same time, may further support malignant progression due to the loss of tumor suppressor genes as e.g. p53.
Subject(s)
Breast Neoplasms/genetics , Chromosomes, Human, Pair 17 , DNA Fragmentation , Chromosome Mapping , Cloning, Molecular , Female , Genetic Markers , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Metaphase , Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
BACKGROUND: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin). The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt) ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3. CONCLUSIONS/SIGNIFICANCE: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.
Subject(s)
Cell Nucleus/metabolism , Drosophila Proteins/metabolism , RNA Splicing/genetics , Animals , Cell Line , Chromatin Immunoprecipitation , Drosophila Proteins/genetics , Drosophila melanogaster , Exosome Multienzyme Ribonuclease Complex , Fluorescent Antibody Technique , Humans , In Situ Hybridization, Fluorescence , Mutation , RNA Interference , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , beta-Globins/geneticsABSTRACT
Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.
Subject(s)
Exosomes/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Protein Precursors/metabolism , Ribonucleoproteins/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Chironomidae/cytology , Chironomidae/genetics , Chironomidae/metabolism , Chromosomes/genetics , Chromosomes/metabolism , Chromosomes/ultrastructure , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Exosomes/ultrastructure , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Immunoprecipitation , Microscopy, Confocal , Microscopy, Immunoelectron , Protein Binding , Protein Precursors/genetics , RNA Interference , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/genetics , Transcription, GeneticABSTRACT
Female mammalian cells inactivate transcription from one of their X chromosomes to equalize gene expression of X-linked genes between males and females. Inactivation is a multistep process that involves a large non-coding RNA termed XIST, a variety of epigenetic modifications of chromatin, and alterations in protein composition such as enrichment of the histone variant macroH2A. We show here that inactive X chromosomes are also enriched in a well-characterized protein component of the nuclear scaffold, SAF-A. This protein has been implicated in chromatin organization, owing to its high specificity for scaffold-associated region (SAR)-DNA, in transcriptional regulation, e.g. of hormone-regulated genes, owing to its functional interaction with steroid receptors, and in RNA processing, owing to its interaction with RNA and heterogeneous nuclear ribonucleoprotein (hnRNP) particles. After near complete removal of DNA and associated chromatin proteins such as macroH2A, SAF-A remains with the "nuclear matrix", still highlighting the former position of inactive X chromosomes. Interestingly, the enrichment of SAF-A in the inactive X chromosome depends on the RNA binding domain of the protein, the RGG box, raising the possibility that interaction of SAF-A with XIST RNA may contribute to the silencing of X-linked genes by local changes in nuclear architecture.
Subject(s)
Chromosomes, Human, X/metabolism , Dosage Compensation, Genetic , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Nuclear Matrix/metabolism , RNA, Untranslated/metabolism , Cells, Cultured , Cloning, Molecular , DNA Primers , Fluorescent Antibody Technique , Humans , Protein Binding , Protein Structure, Tertiary , RNA, Long NoncodingABSTRACT
Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.