Project description:mRNA half-life profiling in the bacterium Caulobacter crescentus was performed in synchronized cells collected from different stages of the cell cycle including swarmer cells, stalk cells (45 min post synchrony), and predivisional cells (90 min post synchrony). For each cell population, transcription was disrupted by the antibiotic rifampicin, and RNA samples were collected at different time points to measure the mRNA half-lives.
Project description:mRNA half-life profiling in the bacterium Caulobacter crescentus was performed in cells that were inhibited in translation initiation (retapamullin) or elongation (chloramphenicol) by shutting of transcription with the antibiotic rifampicin, and following mRNA abundance at 1, 2, 4, 8, and 15 minutes post rifampicin. All RNA measurements were performed on cells grown to mid-log in M2G minimal growth medium. Two biological replicates time coursees were collected from independent starter cultures.
Project description:From synthesis to decay, mRNA changes its protein partners, yet previous studies were limited in that they profiled only the mixed pools of RNA-binding proteins (RBPs) without temporal resolution. Here, we provide time-resolved profiles of human mRNA interactome by combining pulse metabolic labeling, photoactivatable ribonucleoside crosslinking, poly(A) RNA isolation, and mass spectrometry. We captured stage-specific RBPs across 10 time points and quantified over 700 RBPs. The chronological orders of mRNA binding are generally consistent with the known functions and localizations of RBPs: pre-mRNA processing factors are detected as “early binders” while decay factors appear as “late binders”. Notably, stress granule proteins are enriched in “aged” mRNPs, suggesting their roles in the final stage of mRNA life cycle. Many late binders also interact with viral transcripts, implying their regulatory activities on viruses. Furthermore, we built a computational model to systematically identify RBPs with unexpected binding dynamics, which indicates their unknown functions. Our study reveals a dynamic landscape of mRNP remodeling, offering new functional insights into RNA-protein interaction during mRNA life cycle.
Project description:The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiological role, and plays an important role in determining levels of gene expression. We have used a genome wide approach to characterize mRNA decay in Plasmodium falciparum. We found that globally, rates of mRNA decay increase dramatically during the asexual intraerythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 minutes, yet this was extended to an average of 65 minutes during the late schizont stage of development. Thus a major determinant of mRNA decay rate appears to be linked to the stage of intraerythrocytic development. Furthermore, we have found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intraerythrocytic development. This type of genome wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum. Keywords: Plasmodium falciparum treated with actinomycin D
Project description:mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ?100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.
Project description:We have identified NANOS2 bound targets transcriptome wide by employing CRAC and RNA-seq in mouse SSCs. We have determined the average mRNA half-life in SSCs by SLAM-seq. By comparing CRAC and SLAM-seq datasets, we have demonstrated that NANOS2 binding reduces mRNA stability in SSCs.