GSE41785
Global analysis of Upf1 in mESCs reveals expanded scope of nonsense-mediated mRNA decay
Nonsense-mediated mRNA decay (NMD) is a conserved RNA surveillance pathway that is an important modulator of disease pathology and is required for embryonic development.  Despite significant research effort, the rules that govern NMD remain incompletely understood. Here we used a combined¬ approach, integrating RNA-Seq, ribosome footprinting, and CLIP-Seq analysis of the essential NMD factor Upf1, to provide a more complete picture of the role of NMD in modulating gene expression in murine embryonic stem cells (mESCs).  We show that presence of an exon-exon junction =50 nucleotides (nt) downstream of a termination codon (dEJ) contributes to NMD independently of 3'' UTR length, but has stronger effects in genes with shorter 3'' UTRs.  We also map translated upstream open reading frames (uORFs) in mESCs and show that they are associated with NMD regulation, especially of genes encoding transcription factors, and we find that lowly translated mRNAs can escape NMD.  Finally, we identify over 200 direct binding targets of Upf1 and describe a pathway of Upf1-dependent gene regulation reliant on Upf1 binding to the 3'' UTR and independent of presence of a dEJ.   Together, these analyses characterize known and discover novel determinants of NMD and establish a broader role in mESC gene regulation for Upf1. Overall design: mRNA-Seq analysis of wildtype (2 samples), translationally inhibited (by cycloheximide treatment, 2 samples),  control-depleted (2 samples),  and Upf1-depleted (4 samples) mouse embryonic stem cells (mESCs); CLIP-Seq analysis of Upf1 (5 samples, and 5 samples of IgG control CLIP-Seq); Ribosome footprint profiling of wildtype (1 sample), control-depleted (1 sample), and Upf1-depleted (1 sample) mESCs