Cross-linking of mRNA to proteins by irradiation of intact cells with ultraviolet light.

Abstract

Ehrlich ascites tumor cells were irradiated with ultraviolet light to cross-link intracellular RNAs to their tightly bound proteins. The efficiency of such cross-linking in vivo was measured by two independent methods, namely by phenol/chloroform extraction and by isopycnic centrifugation. After short irradiation times more than 70% of the ribosomal RNAs and more than 80% of the mRNA were found to be cross-linked to protein. The size of the cross-linked RNAs was essentially unchanged, indicating that the irradiation does not lead to extensive fragmentation of the RNA. Covalent poly(A)-containing mRNA-protein complexes were isolated by oligo(dT)-cellulose chromatography in the presence of sodium dodecylsulphate. The complexed proteins were freed from RNA by extensive nuclease degradation and analysed by polyacrylamide gel electrophoresis. The results show that some specific proteins are associated with poly(A)-containing RNA in vivo.