Project description:These datasets contain the RNA sequences of three kinds of sample replicate groups obtained from our experiment. They were taken from distal roots and the thickened proximal region, as well as the enhanced thickening of proximal roots in mechanically treated plants. With these RNA sequences, we are profiling the response in Bradypodium distachyon.
Project description:These datasets contain the RNA sequences of three kinds of sample replicate groups obtained from our experiment: the wild type (WT), SZOE, and szAR. They were taken in the time interval of 0, 10, 30 and 60 minutes. With these RNA sequences, we are profiling the touch response in Bradypodium distachyon taken at different times.
Project description:To better resolve the pronounced transcriptional changes observed around temperature transitions, we increased sampling density at the boundaries of the thermocycle.
Project description:The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. Using Bacterial Artificial Chromosome (BAC) transgenic mice which express EGFP-tagged ribosomal protein L10a in defined cell populations, we have developed a methodology to affinity purify polysomal mRNAs from genetically defined cell populations in the brain. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. We find that even two morphologically indistinguishable, intermixed subclasses of medium spiny neuron display vastly different translational profiles and present examples of the physiological significance of such differences. This genetically targeted Translating Ribosome Affinity Purification (TRAP) methodology is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations. Keywords: Cell Type Comparison For each cell type and treatment (cell = D1 or D2, treatment = acute or chronic, saline or cocaine) three independent TRAP replicates were collected, and total RNA from the immunoprecipitates were amplified and hybridized. Data were normalized with the GC-RMA algorithm, and expression values on each chip were normalized to that chip’s 50th percentile. Data were then converted to log2 scale. We recommend that only genes where more than one sample has a normalized intensity larger than 16 (4 in log2 scale) should be kept in the analysis.
Project description:Investigation of proteomic changes in HEK 293T cells after 8h of actinomycin D treatment with special focus on RNA modifying enzymes.
Project description:Cross-linking of living cells followed by mass spectrometry identification of cross-linked peptides (in situ CLMS) is an emerging technology to study protein structures in their native environment. One of the inherent difficulties of this approach is the high complexity of the samples following cell lysis. This difficulty largely limits the identification of cross-links to the more abundant proteins in the cell. Here, we describe a targeted approach in which an antibody pulls a specific protein-of-interest out of the lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the antibody target and its interactors. By using an antibody against the CCT chaperonin, we obtained over two hundred cross-links that provide in situ evidence for the subunit arrangement of CCT and its main interactions with prefoldin. Antibodies against tubulin likewise provided in situ evidence for the structure of the microtubule including the seam. Finally, the approach was also successful in identifying cross-links on a protein expressing at very low amounts (tau in non-neuronal cells). These results demonstrate the general applicability of antibody-based sample simplification for in situ CLMS.
Project description:The intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of αsynuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.
Project description:Formaldehyde is a widely used fixative in biology and medicine. The current chemical model for formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this model. Instead, our data show that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield unusual fragments with a modification of one carbon atom. We apply this new understanding of the underlying cross-linking chemistry to the structural approach of cross-linking coupled to mass spectrometry. First, we cross-linked a mixture of purified proteins with formaldehyde. Our new analysis readily identified tens of cross-links from these proteins, which fit well with their atomic structures. We then perform in-situ cross-linking of human cells in culture. We identified 469 intra-protein and 90 inter-protein cross-links, which also fit well with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. We highlight an example in which formaldehyde cross-links localize the binding site of βNAC on the ribosome. We also find several interactions of actin with auxiliary proteins. Our findings not only expand our understanding of formaldehyde reactivity and toxicity, but also clearly demonstrate how to use this potent reagent for structural studies.