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Cross-linking of mRNA to Proteins by Irradiation of Intact Cells with Ultraviolet Light
Anton J. M. WAGENMAKERS
Laboratorium voor Biochemie, Katholieke Universiteit Nijmegen, Geert-Grooteplein Noord 21, NL-6525-EZ Nijmegen, The Netherlands
Search for more papers by this authorRita J. REINDERS
Laboratorium voor Biochemie, Katholieke Universiteit Nijmegen, Geert-Grooteplein Noord 21, NL-6525-EZ Nijmegen, The Netherlands
Search for more papers by this authorCorresponding Author
Walther J. VAN VENROOIJ
Laboratorium voor Biochemie, Katholieke Universiteit Nijmegen, Geert-Grooteplein Noord 21, NL-6525-EZ Nijmegen, The Netherlands
*To whome correspondence should be addressed.Search for more papers by this authorAnton J. M. WAGENMAKERS
Laboratorium voor Biochemie, Katholieke Universiteit Nijmegen, Geert-Grooteplein Noord 21, NL-6525-EZ Nijmegen, The Netherlands
Search for more papers by this authorRita J. REINDERS
Laboratorium voor Biochemie, Katholieke Universiteit Nijmegen, Geert-Grooteplein Noord 21, NL-6525-EZ Nijmegen, The Netherlands
Search for more papers by this authorCorresponding Author
Walther J. VAN VENROOIJ
Laboratorium voor Biochemie, Katholieke Universiteit Nijmegen, Geert-Grooteplein Noord 21, NL-6525-EZ Nijmegen, The Netherlands
*To whome correspondence should be addressed.Search for more papers by this authorDefinition. Dalton, mass equivalent to 1/12 the mass of the 12C nuclide.
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.
Abbreviations
-
- mRNP
-
- mRNA-protein complex or messenger ribonucleoprotein
-
- poly(A)-rich mRNA
-
- polyadenylated messenger RNA
Enzymes
-
-
- Micrococcal nuclease (EC 3.1.31.1)
-
-
- ribonuclease A (EC 3.1.27.5)
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