ISOLATION AND CHARACTERIZATION OF ENZYMES INVOLVED IN RNA PROCESSING (SPLICING, ADENOVIRUS, LIGASE, BETA-GLOBIN)

Abstract

This thesis describes the isolation and characterization of enzymes that are involved in eukaryotic RNA processing. Chapter I describes the purification of an RNA ligase from HeLa cells that catalyzes the intra- as well as intermolecular ligation of linear RNA substrates possessing 5'-hydroxyl and 2',3'-cyclic phosphate termini in the presence of ATP or dATP. In this reaction, the 2',3'-cyclic phosphate is incorporated into a 3',5'-phosphodiester bond. The activity of the purified enzyme is dependent on the addition of ATP or dATP, a divalent cation (Mg('+2)), and 5'-hydroxyl, 2',3'-cyclic phosphate-terminated RNA substrates. No ligation occurs with the substrates (,OH)(UP)(,10)G(3') or (,OH)(UP)(,10)G(2')p. The role of this enzyme in vivo remains unknown. Chapter II describes the isolation of three fractions (Ia, Ib and II) from a HeLa cell nuclear extract that are required for splicing both Adenovirus precursor messenger RNA (pre-mRNA) and (beta)-globin pre-mRNA. The combination of fractions Ib and II, in the presence of ATP and pre-mRNA, catalyzes cleavage at the 5' splice site, releasing the first exon, and formation of an intron-exon lariat RNA molecule. If fraction Ia is included in the reaction, mature RNA is formed, and the intron is released in a lariat configuration. The activity in fraction II is abolished by treatment with micrococcal nuclease, whereas the other fractions are unaffected by this treatment. The fractions have been distinguished by their sensitivities to N-ethylmaleimide and heat treatment. Sedimentation of the standard splicing reaction containing labeled pre-mRNA and isolated fractions through sucrose gradients reveals the presence of fast sedimenting complexes that contain spliced RNA and putative reaction intermediates. The involvement of these complexes in splicing is suggested by the fact that they are ATP-dependent and are not formed when mutant pre-mRNAs not capable of being spliced are used as substrates.