Isolation and characterization of two enzymatic activities involved in RNA metabolism

Abstract

The work presented in this thesis describes the isolation and characterization of an RNA cyclase and a RNA debranching enzyme from Hela cell extracts.;A. RNA cyclase catalyzes the conversion of 3{dollar}\sp\prime{dollar}-phosphate ends of RNA chains to the 2{dollar}\sp\prime{dollar},3{dollar}\sp\prime{dollar}-cyclic phosphate derivative. We have purified this activity from the cytosolic fraction from HeLa cells approximately 6000 fold. The formation of 1 mol of 2{dollar}\sp\prime{dollar},3{dollar}\sp\prime{dollar}-cyclic ends is associated with the hydrolysis of 1 mol of ATP to AMP and PPi.;This enzyme is likely to be involved in tRNA splicing in animal cells providing the 2{dollar}\sp\prime{dollar},3{dollar}\sp\prime{dollar}-cyclic phosphate end required by the HeLa RNA ligase.;B. The branched RNA molecules, RNA lariats, produced during the splicing of nuclear mRNA precursors are generated by the formation of a 2{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} phosphodiester bond between the 5{dollar}\sp\prime{dollar}-phosphate end of the intron and the 2{dollar}\sp\prime{dollar}-hydroxyl moiety of a specific A residue near its 3{dollar}\sp\prime{dollar} end. The 2{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} phosphodiester bonds of the branch are resistant to the action of various known RNases. An RNA debranching activity have been purified 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the specific hydrolysis of the 2{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} phosphodiester bond of branched RNA yielding a 2{dollar}\sp\prime{dollar}-hydroxyl group and a 5{dollar}\sp\prime{dollar}-phosphate end. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. This fraction is a valuable reagent for the identification of lariat molecules among the splicing products.