In vivo cross-linking of proteins to mRNA in human cells

Human KB cells were irradiated with ultraviolet light to cross-link mRNA to its associated proteins. More than 75% of both the poly(A)-containing and the poly(A)-lacking mRNAs were cross-linked to proteins after 3 min irradiation. Glycerol gradient analysis showed that no significant RNA chain breakage occurred during this treatment. Cross-linked poly(A)-containing mRNA-protein complexes were purified by oligo(dT)cellulose chromatography in the presence of sodium dodecylsulphate. CsCl gradient analysis revealed that the low salt eluted particles had a buoyant density of about 1.47 g/cm3. To determine which proteins were cross-linked to mRNA, covalent mRNA-protein complexes, labeled in their RNA moiety, were exhaustively treated with nucleases. Polyacrylamide gel analysis showed that most of the residual RNA-radioactivity was covalently bound to proteins of 73000, 69000 and 52000 molecular weight.