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 mRNAprotein complex (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 in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription -export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription -coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.