Project description:The goal of this study is to identify ribosome-protected mRNA fragments (RPFs) to investigate gene expression dynamics at both transcriptional and translational levels associated with NAT10 in mouse hearts
Project description:To identify potential mRNAs that are acetylated by NAT10, RIP with a NAT10 antibody and acRIP with a ac4C antibody were conducted in DLD-1 and SW480 cells
Project description:The goal of this study is to investigate how NAT10 regulates heart development in mice. mRNA profiles in hearts of Nat10flox/flox and cardiomyocyte-specific Nat10 knockout (Nat10-CKO) mice at 10 days old were generated by deep sequencing using Illumina novaseq x plus (n=3 for each group).
Project description:Having established NAT10’s regulatory role in lysine 2-hydroxyisobutyrylation, we aimed to identify the histone Khib sites targetedby NAT10. To this end, we extracted and digested core histones from cells with or without NAT10 depletion, followed by chemical derivatization of amine groups. The resulting peptides were analyzed by data-independent acquisition (DIA) mass spectrometry to detect the NAT10-modulated Khib sites.
Project description:The sonication-resistant heterochromatin (srHC) fragments of mESCs were isolated by sucrose gradient ultracentrifugation, and the heterochromatin-associated proteins were enriched by Co-IP combined with mass spectrometry (Co-IP/MS) using anti-H3K9me3, IgG was used as a negative control. The Nat10 interacted proteins in mESCs were isolated by 3×Flag-Nat10 fusion protein overexpression and performed IP-MS using anti-Flag, IgG antibody was used as a negative control.
Project description:Massive numbers of modified bases in mRNAs sculpt the epitranscriptome and play vital roles in RNA metabolism. The only known acetylated RNA modification, N-4-acetylcytidine (ac4C), is highly conserved across cell types and among species. Although the GCN5-related acetyltransferase 10 (NAT10) functions as an ac4C writer, the mechanism underlying the acetylation process is largely unknown. In this study, we identified the NAT10/PCBP/TDP43 complex as an mRNA ac4C writer in mammalian cells. We identified RNA-binding proteins (RBPs) affiliated with two different families, PCBP1/2 (poly(rC)-binding protein 1/2) and TDP43 (TAR DNA binding protein 43), as NAT10 adaptors for mRNA tethering and substrate selection. Knockdown of the adaptors resulted in decreased mRNA acetylation abundance in HEK293T cells, with globally reduced density in 5`-untranslated region (UTR) and coding sequence (CDS) and ablated cytidine-rich ac4C motifs. The adaptors also affect the ac4C sites by recruiting NAT10 to their binding sequences. The presence of the NAT10/PCBP/TDP43 complex in mouse testes highlights its potential physiological functions in vivo. These findings reveal the composition of the mRNA ac4C writer complex in mammalian cells and expand our knowledge of mRNA acetylation and ac4C site preferences.
Project description:Massive numbers of modified bases in mRNAs sculpt the epitranscriptome and play vital roles in RNA metabolism. The only known acetylated RNA modification, N-4-acetylcytidine (ac4C), is highly conserved across cell types and among species. Although the GCN5-related acetyltransferase 10 (NAT10) functions as an ac4C writer, the mechanism underlying the acetylation process is largely unknown. In this study, we identified the NAT10/PCBP/TDP43 complex as an mRNA ac4C writer in mammalian cells. We identified RNA-binding proteins (RBPs) affiliated with two different families, PCBP1/2 (poly(rC)-binding protein 1/2) and TDP43 (TAR DNA binding protein 43), as NAT10 adaptors for mRNA tethering and substrate selection. Knockdown of the adaptors resulted in decreased mRNA acetylation abundance in HEK293T cells, with globally reduced density in 5`-untranslated region (UTR) and coding sequence (CDS) and ablated cytidine-rich ac4C motifs. The adaptors also affect the ac4C sites by recruiting NAT10 to their binding sequences. The presence of the NAT10/PCBP/TDP43 complex in mouse testes highlights its potential physiological functions in vivo. These findings reveal the composition of the mRNA ac4C writer complex in mammalian cells and expand our knowledge of mRNA acetylation and ac4C site preferences.