Solid-phase processing of U2 snRNA precursors.
HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component.
D000906:Antibodies; D000936:Antigen-Antibody Complex; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D012322:RNA Precursors; D012342:RNA, Small Nuclear / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007202:Indicators and Reagents; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear