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.
D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006801:Humans; D008970:Molecular Weight; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D012333:RNA, Messenger; D011061:Poly A