ABSTRACT
Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.
Subject(s)
Genomic Instability , Micronuclei, Chromosome-Defective , Animals , Humans , Mice , Chromosomes/genetics , DNA Damage , Genomic Instability/genetics , Phenotype , Sirtuin 1 , Synthetic Lethal MutationsABSTRACT
Islet-brain1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein that organizes the JNK, MKK7, and MLK1 to allow signaling specificity. Targeted disruption of the gene MAPK8IP1 encoding IB1/JIP-1 in mice led to embryonic death prior to blastocyst implantation. In culture, no IB1/JIP-1(-/-) embryos were identified indicating that accelerated cell death occurred during the first cell cycles. IB1/JIP-1 expression was detected in unfertilized oocytes, in spermatozoa, and in different stages of embryo development. Thus, despite the maternal and paternal transmission of the IB1/JIP-1 protein, early transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocytes.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/physiology , Nuclear Proteins/physiology , Trans-Activators/physiology , Animals , Apoptosis , Blastocyst/metabolism , Blotting, Western , Cell Death , Cell Division , Cell Survival , DNA, Complementary/metabolism , Embryo, Mammalian/metabolism , Female , Heterozygote , MAP Kinase Kinase 7 , MAP Kinase Kinase Kinases/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Spermatozoa/metabolism , Testis/metabolism , Transfection , Zygote/metabolismSubject(s)
Receptors, Transferrin/biosynthesis , Animals , Chickens/genetics , DNA/genetics , DNA Probes , Deferoxamine/pharmacology , Feedback , Gene Expression Regulation/drug effects , Humans , Iron/pharmacology , Iron-Regulatory Proteins , Mice , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , RNA, Messenger/biosynthesis , RNA-Binding Proteins/pharmacology , Receptors, Transferrin/geneticsABSTRACT
Transcripts from the Dd ras gene can only be detected once starved cells have begun to aggregate (Reymond et al., Cell 39: 141-148, 1984). We show in this report that the three transcripts which originate from Dd ras during normal development differ in their 5' ends. In suspension of starved single cells, one major Dd ras RNA accumulates upon addition of cAMP. It seems that the cAMP regulation of Dd ras expression happens both at the transcriptional and post-transcriptional level. An RNA secondary structure present in the 5' untranslated region of the gene is proposed to be important in this post-transcriptional regulation.
Subject(s)
Dictyostelium/genetics , Gene Expression Regulation, Fungal , Genes, ras , Transcription, Genetic , Base Sequence , Cyclic AMP/metabolism , Dictyostelium/growth & development , Endoribonucleases/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Fungal , RNA, Messenger , Restriction Mapping , Ribonuclease HABSTRACT
We describe a simple method for the affinity purification of specific RNA-binding proteins. DNA sequences corresponding to the protein-binding site of the RNA are subcloned into an in vitro transcription vector between the T7 viral promoter and a poly(A) track. A polyadenylated RNA transcript is bound to poly(U)-Sepharose and subsequently incubated with a cellular extract prepurified on heparin-agarose. Specifically adsorbed proteins are recovered in high yield and purity from the affinity matrix by high salt elution. Using this method we isolated the iron regulatory factor (IRF), a cytoplasmic protein which binds to specific palindromic elements in the 5' and 3' untranslated sequences of ferritin and transferrin receptor mRNA, respectively. Activation and binding of this regulatory factor correlates with increased transferrin receptor mRNA stability and inhibition of ferritin translation. The purified factor from human placenta migrates as a monomer in gel chromatography, but is present in equimolar amounts of two proteins with molecular weights of 95 and 100 kDa when analysed by SDS/PAGE. The two proteins are highly related as judged by the identity of their isoelectric points and their specificity to form RNA-protein complexes.
Subject(s)
Carrier Proteins/isolation & purification , Iron/metabolism , Placenta/analysis , Base Sequence , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Humans , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , RNA-Binding ProteinsABSTRACT
The murine histocompatibility class I genes, H-2 Kb and Kk, display considerable homology at their 3' ends. In fact, from exon 5 to the termination codon, only two nucleotides differ between the two genes, one at the 5' end and the other at the 3' end of intron 7. Despite this similarity, the gene products have distinctly different mol. wts as determined by SDS-PAGE. By constructing two hybrid genes, pC2 and pC4, we demonstrated that it is the cytoplasmic parts of the antigens (encoded by exons 6-8) which are responsible for the major difference in mol. wt. We have used site-directed mutagenesis to change the two nucleotides in intron 7 of the H-2 Kk gene to those present in the H-2 Kb gene. S1 nuclease mapping has been used to identify the actual splice site of the authentic Kb and Kk genes, the hybrid genes and the mutagenized genes. We have shown that it is the 3' nucleotide difference, nine nucleotides upstream of the 3' splice site, which causes the different excision of intron 7 of the Kb gene. The 5' nucleotide difference does not alter the splicing. The choice of branch points and 3' splice signals for intron 7 of five H-2 class I genes, is discussed.
Subject(s)
Genes , H-2 Antigens/genetics , Major Histocompatibility Complex , RNA Splicing , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA Restriction Enzymes , Electrophoresis, Polyacrylamide Gel , H-2 Antigens/isolation & purification , Mice , Molecular Weight , Sequence Homology, Nucleic AcidABSTRACT
The presence of terminal deoxynucleotidyl transferase (TdT) has been determined in neoplastic cells from 50 patients with non-hematologic tumors as well as neoplastic cells from 85 patients with hematologic malignancies. The results indicate that TdT is not present in cells from non-hematologic tumors, Hodgkin's lymphoma, B cell lymphoproliferative disorders, peripheral T cell neoplasms, reactive lymphadenopathy, and acute non-lymphocytic leukemia. In contrast, TdT activity is present in non-T non-B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, T cell lymphoblastic lymphoma and chronic granulocytic leukemia in blast crisis. It is concluded that the TdT assay is a measurement useful in the differential diagnosis of some hematologic malignancies.
