title: Purification and characterization of a novel mammalian endoribonuclease.
abstract: Endonuclease-mediated mRNA decay appears to be a common mode of mRNA degradation in mammalian cells, but yet only a few mRNA endonucleases have been described. Here, we report the existence of a second mammalian endonuclease that is capable of cleaving c-myc mRNA within the coding region in vitro. This study describes the partial purification and biochemical characterization of this enzyme. Five major proteins of approximately 10-35 kDa size co-purified with the endonuclease activity, a finding supported by gel filtration and glycerol gradient centrifugation analysis. The enzyme is an RNA-specific endonuclease that degrades single-stranded RNA, but not double-stranded RNA, DNA or DNA-RNA duplexes. It preferentially cleaves RNA in between the pyrimidine and purine dinucleotides UA, UG, and CA, at the coding region determinant (CRD) of c-myc RNA. The enzyme generates products with a 3'hydroxyl group, and it appears to be a protein-only endonuclease. It does not possess RNase A-like activity. The enzyme is capable of cleaving RNAs other than c-myc CRD RNA in vitro. It is Mg(2+)-independent and is resistant to EDTA. The endonuclease is inactivated at and above 70 degrees C. These properties distinguished the enzyme from other previously described vertebrate endonucleases.
authors.affiliation: Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada.
authors.forename: Kirk
authors.initials: K
authors.lastname: Bergstrom
authors.forename: Joel C
authors.initials: JC
authors.lastname: Urquhart
authors.forename: Alaeddin
authors.initials: A
authors.lastname: Tafech
authors.forename: Erin
authors.initials: E
authors.lastname: Doyle
authors.forename: Chow H
authors.initials: CH
authors.lastname: Lee
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D016259:Genes, myc / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008322:Mammals; D016366:Open Reading Frames / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D004722:Endoribonucleases; D008274:Magnesium