Project description:This SuperSeries is composed of the following subset Series: GSE31220: Polysome-associated mRNA levels upon glucose repletion GSE31392: Timecourse of total and polysome-associated mRNA levels post glucose deprivation Refer to individual Series
Project description:Characterization of the riboproteome composition in quiescent cells and post-translational reactivation. To characterize ribosome heterogeneity during the exit from quiescence, the protein composition of ribosomal particles from stationary phase and nutrient-stimulated cells was assessed. A label-free quantitative mass spectrometry strategy was taken to compare quiescent yeast cells with cells that had been nutrient stimulated for 30 and 60 min. To this end, crude extracts from these cells were subjected to sucrose gradient centrifugation and three fractions free (F); monosome (M=80S + 60S + 40S) and polysome (P) were analyzed by nano-HPLC-MS/MS. A total of 528 proteins were identified.
Project description:Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2’-O-methylguanosine, 2’-O-methylcytidine, N4-acetylcytidine and 5-formylcytidine), and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs, Rra1. Additionally, we find that mRNA modification levels change in response to heat shock, glucose starvation and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs, and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.
Project description:The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae. Experimental benchmark of five different protocols (3tfill, bpmI, internal, rnaseq and yoon) for genome-wide identification of polyadenylation sites in Saccharomyces cerevisiae and transcript quantification. RNA was extracted from WT cells grown in glucose (ypd) or galactose (ypgal) as carbon source. The same RNA was used for 3 independent library constructions (technical replicates, rep).
Project description:Dynamic 3' UTRs variation mediated by APA is one of the major determinants of post-transcriptional regulation. Though some progress has been made in understanding its function in mammalian cells, the role of APA in the model organism S. cerevisiae, which lack of miRNAs, is a matter of debate. Unlike mammalian cells, most of S. cerevisiae genes tend to express mRNAs with longer 3′ UTRs in proliferating cells. Further analysis demonstrated that 3' UTRs length and mRNA expression were negatively correlated in different growth conditions. By combining APA sequencing and polysome profiling, we observed that mRNA isoforms with shorter 3' UTRs with a higher translational efficiency in proliferating cells but not quiescent. Further analysis demonstrated that different function genes translational efficiency control by APA are differential modulated and closely related with growth conditions. Furthermore, we observed the correlation between asRNAs and translational efficiency of different length 3′ UTR transcripts, suggests asRNAs may involve in the regulation of translational efficiency mediated by APA. Thus, this study indicates conservation of molecular function of APA in different Eukaryotes, highlighting the importance of APA in regulating gene function.