Project description:N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is crucial for mRNA stability and translation efficiency, yet the underlying function in mammalian preimplantation embryos remains unclear.
Project description:NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. To explore the transcriptome-wide profile of ac4C modification, we mapped the locations of ac4C modification on wild-type (WT) hESCs and NAT10 KD hESCs by NaCNBH3-based chemical ac4C sequencing (ac4C-seq).
Project description:NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. To explore the transcriptome-wide profile of ac4C modification, we mapped the locations of ac4C modification on wild-type (WT) hESCs and NAT10 KD hESCs by high-throughput ac4C RNA immunoprecipitation sequencing (ac4C-RIP-seq).
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:NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. To investigate the effect of NAT10-mediated ac4C acetylation modification on gene expression level, we performed high throughput RNA sequencing experiments of control and NAT10 KD hESCs each with 4 replicates.
Project description:NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. To investigate the effect of NAT10-mediated ac4C acetylation modification on RNA stability at the transcriptome scale, we performed high throughput RNA sequencing experiments of control and NAT10 KD hESCs at three time points each with 3 replicates.
Project description:N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification, present on tRNA, rRNA and recently investigated in eukaryotic mRNA. We report ac4C-seq, a chemical genomic method for single-nucleotide resolution, transcriptome-wide quantitative mapping of ac4C. While we did not find detectable ac4C sites in human and yeast mRNAs, ac4C was induced via ectopic overexpression of eukaryotic acetyltransferase complexes, invariably at a conserved sequence motif. In contrast, cross-evolutionary profiling reveals unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, ncRNA and mRNA from hyperthermophilic archaea. Ac4C is dramatically induced in response to temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Cryo-EM visualization of WT and acetyltransferase-deficient archaeal ribosomes furnishes structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for unravelling this modification’s role in biology and disease.
Project description:How plants control the transition to flowering in response to ambient temperature is only beginning to be understood. In Arabidopsis thaliana, the MADS-box transcription factor genes FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) have key roles in this process. FLM is subject to temperature-dependent alternative splicing, producing two splice variants, FLM-β and FLM-δ, which compete for interaction with the floral repressor SVP. The SVP/FLM-β complex is predominately formed at low temperatures and prevents precocious flowering. In contrast, the competing SVP FLM-δ complex is impaired in DNA binding and acts as a dominant negative activator of flowering at higher temperatures. Our results demonstrate the importance of temperature-dependent alternative splicing in modulating the timing of the floral transition in response to environmental change.