Purification of a RNA debranching activity from HeLa cells.
The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.
D002413:Cations, Divalent; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D012322:RNA Precursors; D012326:RNA Splicing; D000079962:RNA, Circular; D012333:RNA, Messenger / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D000079962:RNA, Circular; D012333:RNA, Messenger; D012313:RNA; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme