Project description:mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells. 293T cells and k562 cells were infected with ORFome library to decay exodogenous gene mRNA decay. Stable infected cells were treated with actinmycin D at 6 well plate and samples were collected in duplicate every hour 0-6h for RNA-seq
Project description:mRNA endogenous decay profiles approach in 293T, HeLa and RPE cells. mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.
Project description:Transcriptional profiling of Human Embryonic Kidney(293T) Cells comparing control untreated 293T cells with 293T cells transfected with A) pcDNA 3.0(-) vector[Invitrogen] (Mock) and B) Expression vector pcDNA 3.0(-) containing cloned Influenza virus H5N1and H11N1-NS1 (Non-Structural1) gene.
Project description:The K562 cell line was obtained from ATCC (American Type Culture Collection, Manassas,VA). And the K562 cell line was transduced using a highly efficient retroviral vector coding HLA-A*11:01. The vectors were transfected into a 293T packaging cell line and replication-defective virus supernatants were harvested. After infection of K562 cells with the supernatant, antibody-directed flow cytometry sorting was done to obtain high expression for HLA-A*11:01. Cells were grown in T75 flasks to a density of 1e9 cells before purification of HLA-I peptides for MS experiments.
Project description:Methylome data obtained from human embryonic kindey (HEK)-293T cells expressing a GFP (293T-GFP) or a truncated form of Arabidopsis DEMETER (DME) 5-methylcytosine (5mC) DNA glycosylase (293T-DMEΔ) analyzed on a Human Methylation 450K BeadChip platform (Illumina). These methylation array data revealed genome-wide DNA methylation patterns of the 293T-GFP cells (without direct 5mC excision activity) and 293T-DMEΔ cells (with artificially implemented direct 5mC excision activity).
Project description:We used microarray to determine the genes whose expression was changed at three or six hours after Y27632 treatment. Three-condition experiment; 293T control vs 293T treated with Y27632 for three hours, 293T control vs 293T treated with Y27632 for six hours.