Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle

mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNP remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA-binding proteins (RBPs) across ten distinct time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed numerous RBPs with unexpected dynamics: the TRanscription-EXport complex (TREX) recruited posttranscriptionally after NXF1 binding, challenging the current view of transcription-coupled mRNA export mechanism, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with GO annotations. Our data can be explored at chronology.rna.snu.ac.kr. Overall design: Pulse-chased mRNA analysis using 10 min 4sU labeling and MTS-biotin pulldown