SLAM-seq for K562 endogenous mRNA decay

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. Overall design: SLAMseq Kinetics Kit - Catabolic Kinetics Module) from Lexogen was performed. Basically, k562 cells were feed with 100uM 4sU for 24h, fresh media containing s4U was changed every 3h. For chasing, old media are removed and cells were washed with PBS three times before adding media without s4U, but 10mM UTP instead, samples were collected at 0, 2, 4, 6h in triplicates for RNA extract and IAA treatment. All the operations were done in red light source to protect 4sU from crosslink. QuantSeq 3'mRNA-seq library were generated and sequenced. The SLAM-seq data was processed with the slam dunk pipeline (https://github.com/t-neumann/slamdunk 0.3.3.) The reads were aligned to the human genome GRCh38. The half-life was estimated according to the SLAM-seq paper; for downstream analyses a cut-off of p-value < 0.05 was applied to the decay rate estimates. Comparing mRNA levels using QuantSeq and RNA-seq poly-A selection.

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Wu Q, Medina SG, Kushawah G, DeVore ML, Castellano LA, Hand JM, Wright M, Bazzini AA