Project description:We combined RNA metabolic labeling and alkylation with droplet-based sequencing to detect newly synthesized mRNAs in single cells. With the classification of labeled and unlabeled precursor and mature mRNAs, we modeled and analyzed the time-dependent RNA kinetic rates associated with the cell cycle. We found both transcription and degradation rates are highly dynamic over the cell cycle and different kinetic regulation types were observed for cycling genes.
Project description:T cell receptor(TCR) engagement in the absence of costimulation leads to a state of T cell tolerance known as anergy. Anergy induction requires new protein synthesis since it is inhibited by cycloheximide. In this experiment, we tried to figure out kinetic properties of the gene expression in anergy induction phase and the subsequent anergy maintenance phase.
Project description:Proteins play a central role in most biological processes within the cell and deciphering how they interact is key to understand their function. Cross-linking coupled to mass spectrometry is an essential tool for elucidating protein-protein interactions. Despite its importance, we still know surprisingly little about the principles that underlie the process of chemical cross-link formation itself and how it is influenced by different physicochemical factors. To understand the molecular details of cross-link formation, we have set-up a comprehensive kinetic model and carried out simulations of protein cross-linking on large protein complexes. We dissect the contribution on the cross-link yield of parameters such as amino acid reactivity, cross-linker concentration, and hydrolysis rate. Our model can compute cross-link formation based solely on the structure of a protein complex, thereby enabling realistic predictions for a diverse set of systems. We quantitatively show how cross-links and mono-links are in direct competition and how the hydrolysis rate and abundance of cross-linker and proteins directly influence their relative formation. We show how cross-links and mono-links exist in a “all-against-all” competition due to their simultaneous formation, resulting in a non-intuitive network of interdependence. We show that this interdependence is locally confined and mainly limited to direct neighbors or residues in direct vicinity. These results enable us to identify the optimal cross-linker concentration at which the maximal number of cross-links are formed. Taken together, our study establishes a comprehensive kinetic model to quantitatively describe cross-link formation for protein-protein interactions.
Project description:Cell-to-cell heterogeneity in gene expression can even be observed in the same type of cells, present in a similar environment. Transcriptional bursting is thought to be one of contributing factors to the heterogeneity, but it remains elusive how the kinetic properties of transcriptional bursting (e.g. burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. To unbiasedly identify genes regulating the kinetic properties of transcriptional bursting, we performed large-scale CRISPR/-Cas9 based screening and functional analysis, and found that Akt/MAPK signaling pathways are involved in the regulation of the kinetic properties of transcriptional bursting.
Project description:RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic χCRAC, a UV cross-linking method that enabled us to quantitatively measure the dynamics of protein-RNA interactions in vivo on a minute time-scale. Here, using kinetic χCRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurement reveal rapid changes in protein-RNA interactions within one minute following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. Kinetic χCRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein-RNA interactions.
Project description:T cell receptor(TCR) engagement in the absence of costimulation leads to a state of T cell tolerance known as anergy. Anergy induction requires new protein synthesis since it is inhibited by cycloheximide. In this experiment, we tried to figure out kinetic properties of the gene expression in anergy induction phase and the subsequent anergy maintenance phase. A.E7 cell, a murine CD4(+) T cell clone, was stimulated with anti-TCR antibody for 0, 2, 4, and 6hrs followed by RNA extraction. For the maintenance phase, A.E7 was stimulated for 16hrs and rested for 5days before RNA extraction.
Project description:Singapore grouper iridovirus (SGIV) is the major agent that causes severe iridovirus diseases in grouper maricluture. Based on the genomic information, a DNA microarray, containing probes corresponding to 162 putative SGIV open reading frames (ORFs) was constucted. The viral microarrays wereused to classify the majority of SGIV transcripts into three temporal kinetic classes (immediate-early, IE; early, E; late, L) during an in vitro infection by their dependence on de novo protein synthesis inhibitor and viral DNA replication. Keywords: drug response