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Updated: Dec 28, 2025

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
Published on: May 3, 2014
An "external" RNA removable from mammalian cells by mild proteolysis
Abstract:
Brief exposure of Chinese hamster ovary cell monolayers prelabeled with [(32)P]phosphate and [(3)H]leucine to 1 mug/ml of trypsin under conditions in which cells remain fully viable causes the release of macromolecular (32)P and (3)H. Whereas ribonuclease treatment was found to affect markedly both the (32)P and (3)H radioactivity, Pronase treatment had little effect on the macromolecular (32)P. Treatment of cells prelabeled with [(3)H]glucosamine and [(32)P]phosphate with insolubilized papain also revealed a parallel release of macromolecular glucosamine together with ribonuclease-susceptible macromolecular phosphate. Lactoperoxidase-mediated radioiodination of surface components in cells prelabeled with [(32)P]phosphate revealed electrophoretic comigration between the (125)I and the (32)P that are removed from the cells by mild proteolysis. Growth of the cells in Bt(2)cAMP-testosterone altered the kinetics of release and nature of the macromolecular (32)P liberated by proteolysis.
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Pre-mRNA Processing: Modification of pre-mRNA Ends
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps...

