Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease.
A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.
D000818:Animals; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology; D002642:Chick Embryo; D006224:Cricetinae; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004722:Endoribonucleases; D007668:Kidney; D008970:Molecular Weight; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011402:Pronase; D012313:RNA / Q000378:metabolism; D012322:RNA Precursors; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000378:metabolism*; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology
D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D012335:RNA, Ribosomal; D012313:RNA; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; D011402:Pronase