12 S small nuclear ribonucleoprotein-associated acidic and pyrimidine-specific endoribonuclease from calf thymus and L5178y cells

Abstract 12 S ribonucleoprotein (RNP) particles were separated from a 45 S RNP complex (Bachmann, M., Zahn, R.K. and Muller, W.E.G. (1983) J. Biol. Chem. 258, 7033–7040) isolated from calf thymus and L5178y cells. The particles were determined to be associated with an acidic endoribonuclease (p I 4.1; pH optimum 6.2). the enzyme requires Mg 2+ and is sensitively inhibited by higher NaCl concentrations. The nuclease specifically degrades poly(U) and poly(C) in an endonucleolytic manner; the end-products are 3′-UMP (85%) and 2′,3′-cyclic UMP (12%). Poly(A) strongly inhibits the p I 4.1 endoribonuclease activity. The Michaelis constant (for poly(U)) was determined as 82 μM and the maximal reaction velocity was 0.54 μmol/μg per h. The endoribonuclease is distinguished from the known pyrimidine-specific ribonucleases (pancreatic ribonuclease and endoribonuclease VII) by further criteria, e.g., resistance to thiol reagents, inhibition by EDTA, Mg 2+ requirement, p I and pH optimum. Using the techniques of counterimmunoelectrophoresis and immunoaffinity column chromatography it was shown that the p I 4.1 endoribonuclease-associated 12 S RNP particles display antigenicity to anti-Sm and anti-(U1)-RNP antibodies. An RNA component, isolated from the 12 S-45 S hypercomplex, was identified as U1-snRNA.