Optimizing In Vitro Pre-mRNA 3' Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract.
Eukaryotic RNA processing steps during mRNA maturation present the cell with opportunities for gene expression regulation. One such step is the pre-mRNA 3' cleavage reaction, which defines the downstream end of the 3' untranslated region and, in nearly all mRNA, prepares the message for addition of the poly(A) tail. The in vitro reconstitution of 3' cleavage provides an experimental means to investigate the roles of the various multi-subunit cleavage factors. Anion-exchange chromatography is the simplest procedure for separating the core mammalian cleavage factors. Here we describe a method for optimizing the in vitro reconstitution of 3' cleavage activity from the DEAE-sepharose separated HeLa cleavage factors and show how to ensure, or avoid, dependence on creatine phosphate. Important reaction components needed for optimal processing are discussed. We also provide an optimized procedure for preparing small-scale HeLa nuclear extracts from adherent cells for use in 3' cleavage in vitro.
Department of Chemistry and Biochemistry, The City College of New York, 160 Covent Avenue, New York, NY, 10031, USA.|Biochemistry Ph.D. Program, The City University of New York Graduate Center, New York, NY, 10016, USA.
Mihwa
M
Na
Department of Immunology and Microbial Sciences, The Scripps Research Institute, Jupiter, FL, USA.
Susana T
ST
Valente
Department of Chemistry and Biochemistry, The City College of New York, 160 Covent Avenue, New York, NY, 10031, USA. kr107@sci.ccny.cuny.edu.|Biochemistry Ph.D. Program, The City University of New York Graduate Center, New York, NY, 10016, USA. kr107@sci.ccny.cuny.edu.|Program in Chemistry, The Graduate Center of the City, University of New York, New York, NY, 10016, USA. kr107@sci.ccny.cuny.edu.
Kevin
K
Ryan
D002448:Cell Adhesion; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D006367:HeLa Cells; D006801:Humans; D010725:Phosphocreatine / Q000737:chemistry; D059367:RNA Cleavage; D012322:RNA Precursors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry* / Q000302:isolation & purification; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification
D002457:Cell Extracts; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D010725:Phosphocreatine
3′ end formation; Cleavage and polyadenylation; Pre-mRNA; Pre-mRNA processing