ORFome decay in 293t and k562

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 Overall design: The 96 well format ORFome library was pool together using an automatic robot. First in 20 bins were created based in ORF size, then those 20 were pooled in 5 groups, grown, DNA maxi-preparation done. After quantification, the five groups were mixed into a single cocktail. The ORFome cocktail was transfected with lentivirus vector: Pax2 and VSV-G into 293T cells for virus packing. Media containing virus were filtered and ultracentrifuged to enrich virus for infection. 293T cells and k562 cells were infected and selected with puromycin for a week, at the concentration of 293T(2ug/ml), K562(0.75ug/ml). Then, cells were treated with Actinomycin D (5ug/ml) at 6 well plate and samples were collected in duplicate every hour 0-6h for RNA-seq. Specific oligos were used to target the surrounding barcode region of ORFome to generate library and sequenced.