Anti-SLBP RIPchip data
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE59915 | GEO | 2015/12/16
SECONDARY ACCESSION(S): PRJNA257063
REPOSITORIES: GEO
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Project description:The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. They are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in at a conserved stem-loop (SL) structure. This SL structure interacts stably with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including RNA Binding Protein IP followed by microarray analysis (RIP-ChIP) to analyze the RNA binding landscape of SLBP. Reference based design on two color Agilent DNA microarrays. Experimental sample is labeled in Ch1 (Cy3), which are mRNA bound by the SLBP RNA-binding protein. The reference sample is labeled in Ch2 (Cy5) which are the polyribosomal mRNAs from the supernatant that were not bound by anti-SLBP. Controls included no antibody in the IP (which tends to have non-specific background binding to the beads) and pre-incubation of the SLBP with the antibodies antigenic peptide, which blocks antibody binding to the SLBP.
2015-12-16 | E-GEOD-59915 | biostudies-arrayexpress
Project description:we try to investigate the binding of ALYREF and NXF1 on histone mRNA when the cell treated with indicated siRNAs. ALYREF plays key roles in nuclear export of polyadenylated mRNAs and also modulates their 3' processing, but whether it is involved in regulating RNAs beyond polyadenylated mRNAs is unknown. The replication-dependent (RD) histone mRNAs are not polyadenylated, but end in a stem-loop (SL) structure. Here we demonstrate that ALYREF prevalently binds a region next to the SL on RD histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and ensures this binding. To examine how SLBP KD impact ALYREF distribution on the histone mRNA, we carried out ALYREF iCLIP in control and SLBP KD cells. To investigate the functional consequence for ALYREF binding on histone mRNAs, we isolated polyA+ and polyA- RNAs from control and ALYREF KD cells, and carried out RNA-seq separately.
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Project description:Replication-dependent (RD) histone mRNA are the only non-polyadenylated protein coding transcripts in humans. However, a subset of RD histone genes also expresses polyadenylated mRNA upon terminal differentiation to maintain histone proteins. We now show that even in cycling cells, RD histone genes express some poly(A) mRNA. The H2AFX gene, encoding the DNA damage signalling histone variant H2A.X, expresses both an S-phase-specific SL mRNA ending in the typical stem-loop (SL) structure and substantial amounts of an extended poly(A) mRNA. We show, by the selective removal of either mRNA isoform, that the S-phase-specific H2A.X SL mRNA can generate sufficient histone for deposition onto DNA damage responsive (DDR) chromatin in some cell types. In contrast, when cells have evolved to make predominantly H2A.X poly(A) mRNA they then require de novo H2A.X for efficient DDR. This highlights the importance of differential H2AFX mRNA 3’ end processing for the maintenance of effective DDR
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Project description:During S-phase of the cell cycle production of the core histone proteins is precisely balanced with DNA replication. Metazoan mRNAs encoding replication dependent (RD) histones lack polyA tail normally formed by 3’ end cleavage and coupled polyadenylation of the pre-mRNA. Instead, they undergoes to endonucleolytic cleavage on the 3ʹ side of an RNA hairpin (stem loop) producing mRNA with a 3´-stem loop (SL), which is exported from the nucleus for use in translation. The same endonuclease that is involved in normal protein-coding pre-mRNA cleavage, i.e. cleavage and poyladenylation specificity factor 73 (CPSF73), is proposed to catalyse RD pre-histone mRNA cleavage. Additional factors specific to RD pre-histone mRNA processing, including stem loop binding protein (SLBP) and the U7 small nuclear ribonucleoprotein (U7snRNP) that binds to a histone downstream element (HDE) are thought to be involved in CPSF73 targeting to RD pre-histone mRNA. We report that a different histone specific endonuclease (HSE), which like CPSF73 is a metallo β lactamase (MBL) fold protein, is specific for RD pre-histone mRNA cleavage10,11. Crystallographic and biochemical studies reveal HSE has a di-zinc ion containing active site related to that of CPSF73, but which has distinct overall fold. Notably HSE depletion from cells leads to the production of unprocessed RD pre-histone mRNA due to inefficient 3ʹ end processing. The consequent depletion of core histone proteins correlates with a cell cycle defect due to a delay in entering/progressing through S-phase. HSE thus may represent a new type of S-phase specific cancer target.
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Project description:The replication-dependent canonical histone genes are the only genes found in multicellular organisms whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3’ end. We previously demonstrated that arsenic exposure induces polyadenylation of canonical histone H3.1 mRNA in tissue culture system. We report that arsenic is capable of inducing high levels of H3.1 mRNA polyadenylation and marked loss of the Stem-loop binding protein (SLBP) in vivo, which is critical for canonical histone pre-mRNA processing. Ectopic expression of polyadenylated H3.1 mRNA enhances anchorage-independent cell growth and tumor formation in nude mice. Moreover, polyadenylation of H3.1 mRNA causes transcriptional deregulation, G2/M arrest, chromosome aneuploidy and aberration. Importantly, overexpression of H3.3 attenuates arsenic-induced cell transformation. Through ChIP-Seq and RNA-Seq, we try to decipher if polyadenylated H3.1 mRNA could result in depletion of histone variant H3.3 at critical gene regulatory regions. Our study uncovers polyadenylation of H3.1 and resulting displacement of variant H3.3 as a potential new mechanism for arsenic-induced carcinogenesis.
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Project description:Metazoan histone mRNAs are the only cellular eucaryotic mRNAs that are not polyadenylated, ending instead in a conserved stemloop. SLBP is bound to the 3’ end of histone mRNA and is required for translation of histone mRNA. Their expression is tightly cell-cycle regulated. A major regulatory step is rapid degradation of histone mRNA at the end of S-phase or when DNA synthesis is inhibited in S-phase. 3’hExo, a 3’ to 5’ exonuclease, binds to the SLBP/SL complex, and trims histone mRNA to 3 nts after the stemloop. Together with a terminal uridyl transferase, 3’hExo maintains the length of the histone mRNA during S-phase. 3’hExo is essential for initiating histone mRNA degradation on polyribosomes, initiating degradation into the 3’ side of the stemloop. There is extensive uridylation of degradation intermediates in the 3’ side of the stem when histone mRNA is degraded. Here we knocked out TUT7 and 3’hExo and we show that both modification of histone mRNA during S-phase and degradation of histone mRNA involve the interaction of 3’hExo, and a specific TUTase, TENT3B (TUT7, CHHC6). Knockout of 3’hExo prevents initiation of 3’ to 5’ degradation, stabilizing histone mRNA, while knockout of TUT7 prevents uridylation of the mRNA degradation intermediates slowing the rate of degradation. In synchronized 3’hExo KO cells, histone mRNA degradation is delayed but the histone mRNA is degraded prior to mitosis by a different pathway.
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Project description:In higher eukaryotes, maternally provided gene products drive the initial stages of embryogenesis until the zygotic transcriptional program takes over, a developmental process called the midblastula transition (MBT). In addition to zygotic genome activation, the MBT involves alterations in cell-cycle length and the implementation DNA damage/replication checkpoints that serve to monitor genome integrity. Previous work has shown that mutations affecting histone mRNA metabolism or DNA replication checkpoint factors severely impact developmental progression through the MBT, prompting us to characterize and contrast the transcriptomic impact of these genetic perturbations. In this study, we define gene expression profiles that mark early embryogenesis in Drosophila through transcriptomic analyses of developmentally staged (early syncytial vs late blastoderm) and biochemically fractionated (nuclear vs cytoplasmic) wild-type embryo. We then compare the transcriptomic profiles of loss-of-function mutants of the dChk1/Grapes replication checkpoint kinase and the Stem Loop Binding Protein (SLBP), a key regulator of replication-dependent histone mRNAs. Our analysis of RNA spatial and temporal distribution during embryogenesis offers new insights into the dynamics of early embryogenesis. Moreover, we find that grp and slbp mutant embryos display profound and highly similar defects in gene expression, most strikingly in zygotic genome activation, compromising the transition from a maternal to a zygotic regulation of development.
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Project description:To examine whether ALYREF can facilitate the 3'processing of RD histone mRNA,we isolated and sequenced the polyadenylated and nonpolyadenylated RNAs from control and ALYREF KD cells
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Project description:The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of ~100 copies of a tandemly arrayed 5 kB repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ~200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters (12xPR) does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression. ChIP-qPCR and ChIP-Seq experiments were done on homozygous histone locus (HisC) deletion embryos rescued by the 12 copy promoter replacement (PR) or designer wild type (DWT) arrays on the third chromosome (VK33) to determine CLAMP's interaction with the locus
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