Global donor and acceptor splicing site kinetics in human cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE129635 | GEO | 2019/04/12
REPOSITORIES: GEO
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Project description:RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the 'yield' of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.
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Project description:In this work, we quantified mRNA flow rates between subcellular compartments in mouse embryonic stem cells. Combining metabolic RNA labeling, biochemical cell fractionation and RNA sequencing with mathematical modeling we were able to determine the kinetic rates of nuclear pre-, nuclear mature, cytosolic and membrane mRNA derived from more than 9000 protein-coding genes. For more than 5000 genes, we additionally estimated the transcript elongation rate. For most of the transcripts, mature nuclear half-lives are the longest, suggesting nuclear retention to be the rate-limiting step in the mRNA life cycle. Genes encoding transcription factors and immediate early genes possess fast kinetic rates, specifically a short nuclear half-life. Differentially localized mRNAs exhibit distinct combinations of rate constants, suggesting modular control within subcellular compartments. We show that membrane stability is high for membrane-localized mRNA and that cytosolic stability is high for cytosol-localized mRNA. Genes encoding target signals, such as signal peptides or transmembrane domains, have low cytosolic and high membrane half-lives with only slight differences between target signals. Nuclear-encoded mitochondrial proteins show long nuclear mature half-lives and otherwise similar features of cytoplasmic kinetics that do not resemble co-translational targeting to the mitochondria.
2024-09-17 | GSE252199 | GEO
Project description:In this work, we quantified mRNA flow rates between subcellular compartments in mouse embryonic stem cells. Combining metabolic RNA labeling, biochemical cell fractionation and RNA sequencing with mathematical modeling we were able to determine the kinetic rates of nuclear pre-, nuclear mature, cytosolic and membrane mRNA derived from more than 9000 protein-coding genes. For more than 5000 genes, we additionally estimated the transcript elongation rate. For most of the transcripts, mature nuclear half-lives are the longest, suggesting nuclear retention to be the rate-limiting step in the mRNA life cycle. Genes encoding transcription factors and immediate early genes possess fast kinetic rates, specifically a short nuclear half-life. Differentially localized mRNAs exhibit distinct combinations of rate constants, suggesting modular control within subcellular compartments. We show that membrane stability is high for membrane-localized mRNA and that cytosolic stability is high for cytosol-localized mRNA. Genes encoding target signals, such as signal peptides or transmembrane domains, have low cytosolic and high membrane half-lives with only slight differences between target signals. Nuclear-encoded mitochondrial proteins show long nuclear mature half-lives and otherwise similar features of cytoplasmic kinetics that do not resemble co-translational targeting to the mitochondria.
2024-09-17 | GSE256335 | GEO
Project description:Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.
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Project description:DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes. We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed. Keywords: kinetics, gene expression, microarrays Total RNA were extracted from 5 thymus and 6 cortex of B10D2 mice. For each tissue, equal amounts of each RNA were pooled and 15M-BM-5g were labeled by reverse-transcription and hybridized on neurotransmission microarrays. Three replicates of each experiment have been performed.
2007-09-29 | E-GEOD-7574 | biostudies-arrayexpress
Project description:DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes. We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed. Keywords: kinetics, gene expression, microarrays
2007-09-30 | GSE7574 | GEO
Project description:Molecular materials with π-conjugated donor-acceptor (D-A) and acceptor-donor-acceptor (A-D-A) electronic structures have received significant attention due to their usage in organic photovoltaic materials, in organic light-emitting diodes, and as biological imaging agents. Boron-containing molecular materials have been explored as electron-accepting units in compounds with D-A and A-D-A properties as they often exhibit unique and tunable optoelectronic and redox properties. Here, we utilize Stille cross-coupling chemistry to prepare a series of compounds with boron difluoride hydrazones (BODIHYs) as acceptors and benzene, thiophene, or 9,9-dihexylfluorene as donors. BODIHYs with D-A and A-D-A properties exhibited multiple reversible redox waves, solid-state emission with photoluminescence quantum yields up to 10%, and aggregation-induced emission (AIE). Optical band gaps (or highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) gaps) determined for these compounds (2.02-2.25 eV) agree well with those determined from cyclic voltammetry experiments (2.05-2.42 eV). The optoelectronic properties described herein are rationalized with density functional theory calculations that support the interpretation of the experimental findings. This work provides a foundation of understanding that will allow for the consideration of D-A and A-D-A BODIHYs to be incorporated into applications (e.g., organic electronics) where fine-tuning of band gaps is required.
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Project description:Donor-acceptor [2]catenanes based on cyclobis(paraquat-p-phenylene) as the pi-acceptor ring have been used prominently in the construction of functional molecular devices. We report here their thermodynamically controlled synthesis from isolated pi-donor and pi-acceptor rings under the catalytic influence of tetra butylammonium iodide. The initial nucleophilic attack of iodide ion, which opens up the pi-acceptor ring, is followed by complexation to the pi-donor ring and the subsequent catenation of the pi-donor ring by the pi-acceptor ring [2]catenane. The reaction is general in scope and proceeds in high yields, without giving rise to side-products.
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Project description:The active material of optoelectronic devices must accommodate for contacts which serve to collect or inject the charge carriers. It is the purpose of this work to find out to which extent properties of organic optoelectronic layers change close to metal contacts compared to known properties of bulk materials. Bottom-up fabrication capabilities of model interfaces under ultrahigh vacuum and single-atom low temperature (LT)-STM spectroscopy with density functional theory (DFT) calculations are used to detect the spatial modifications of electronic states such as frontier-orbitals at interfaces. The system under consideration is made of a silver substrate covered with a blend of C60 and ZnPc molecules of a few monolayers. When C60 and ZnPc are separately adsorbed on Ag(111), they show distinct spectroscopic features in STM. However, when C60 is added to the ZnPc monolayer, it shows scanning tunneling spectra similar to ZnPc, revealing a strong interaction of C60 with the ZnPc induced by the substrate. DFT calculations on a model complex confirm the strong hybridization of C60 with ZnPc layer upon adsorption on Ag(111), thus highlighting the role of boundary layers where the donor-acceptor character is strongly perturbed. The calculation also reveals a significant charge transfer from the Ag to the complex that is likely responsible for a downward shift of the molecular LUMO in agreement with the experiment.
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Project description:The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ~10 h. Furthermore, repair of the DSBs tends to be error-prone. Both classical and microhomology-mediated end-joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci, and indicates that Cas9-induced DSBs are repaired in an unusual manner.
2018-05-01 | GSE113129 | GEO