Purification and characterization of a novel mammalian endoribonuclease.

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.