Eukaryotic mRNA is bound by a wide array of regulatory proteins, but until recently the full complement of the mRNA-bound proteome has not been systematically characterized. Baltz et al. now use photoreactive nucleoside analogs to cross-link proteins and RNA in human embryonic kidney cells and purify the complexes by PolyA-based precipitation. The authors identify close to 800 mRNA-bound proteins using high-resolution quantitative mass spectrometry, one third of which have not been described as RNA binding before and about 15% of which would not have been predicted to interact with RNA. Among the newly identified mRNA binders are proteins that were previously exclusively annotated as transcription factors (e.g., JUN or NXF1). Baltz et al. also generate the first transcriptome-wide protein occupation map using the previously published PAR-Clip method. The protein-binding sites have a substantially lower single-nucleotide polymorphism frequency than neighboring sites, suggesting they are under high selection pressure in evolution. Several of the single-nucleotide polymorphisms found in mRNA-protein interaction sites have been associated with disease, lending further support to the idea they are functionally important. (Mol. Cell 46, 674–690, 2012)