Project description:Wild-type and RBMX-knockdown HEK293 cells where profiled at the transcriptome and translatome levels for the expression of 50 homeotic genes. Targeted RNA-seq libraries were built and sequenced on an Illumina MiSeq machine to allow for accurate gene expression quantification of this specific set of genes. To assess the binding of RBMX to the same set of mRNAs, RNA immunoprecipitation was also performed on HIS-HA tagged RBMX expressing HEK293 cells.
Project description:Even though proteins are produced from mRNA, the correlation between mRNA levels and protein abundances is moderate in most studies, occasionally attributed to complex post-transcriptional regulation. To address this, we generated a paired transcriptome/proteome time course dataset with 14 time points during Drosophila embryogenesis. Despite a limited mRNA-protein correlation (ρ = 0.54), mathematical models describing protein translation and degradation explain 84% of protein time-courses based on the measured mRNA dynamics without assuming complex post-transcriptional regulation, and allow for classification of most proteins into four distinct regulatory scenarios. By performing an in-depth characterization of the putatively post-transcriptionally regulated genes, we postulated that the RNA-binding protein Hrb98DE is involved in post-transcriptional control of sugar metabolism in early embryogenesis and partially validated this hypothesis using Hrb98DE knockdown. In summary, we present a systems biology framework for the identification of post-transcriptional gene regulation for large-scale time-resolved transcriptome and proteome data.
Project description:The cell division cycle of the unicellular eukaryote Trypanosome brucei is tightly regulated despite the paucity of transcriptional control that results from the arrangement of genes in polycistronic units and lack of dynamically regulated transcription factors. To identify the contribution of dynamic phosphorylation to T. brucei cell cycle control we have combined cell cycle synchronisation by centrifugal elutriation with SILAC quantitative phosphoproteomic analysis. Cell cycle regulated changes in phosphorylation site abundance (917 sites, average 5-fold change) were more widespread and of a larger magnitude than changes in protein abundance (443 proteins, average 2-fold change) and were mostly independent of each other. Hierarchical clustering of co-regulated phosphorylation sites according to their cell cycle profile revealed a bulk increase in phosphorylation occurs across the cell cycle, with a significant enrichment of known cell cycle regulators and RNA binding proteins (RBPs) within the largest clusters. Cell cycle regulated changes in essential cell cycle kinases is temporally co-ordinated with differential phosphorylation of components of the kinetochore and eukaryotic initiation factors, along with many RBPs not previously linked to the cell cycle such as eight PSP1-C terminal domain containing proteins. The temporal profiles demonstrate the importance of dynamic phosphorylation in co-ordinating progression through the cell cycle, and provides evidence that RBPs play a central role in post-transcriptional gene regulation of the T. brucei cell cycle.