Purification and characterization of a novel mammalian endoribonuclease
Purification and characterization of a novel mammalian endoribonuclease
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DOI：
10.1002/jcb.20726
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发表时间：
2006-06-01
影响因子：
4
通讯作者：
Lee, Chow H.
中科院分区：
文献类型：
--
作者：
Bergstrom, Kirk;Urquhart, Joel C.;Lee, Chow H.
核酸内切酶介导的mRNA降解似乎是哺乳动物细胞中mRNA降解的常见模式，但只有少数mRNA核酸内切酶已被描述。在这里，我们报告存在的第二种哺乳动物核酸内切酶，能够在体外切割编码区内的c-myc mRNA。本研究描述了该酶的部分纯化和生化特性。凝胶过滤和甘油梯度离心分析支持了这一发现。该酶是RNA特异性内切核酸酶，其降解单链RNA，但不降解双链RNA、DNA或DNA-RNA双链体。在c-myc RNA的编码区决定簇（CRD）处，它优先切割嘧啶和嘌呤二核苷酸UA、UG和CA之间的RNA。该酶产生具有3 '羟基基团的产物，并且它似乎是仅蛋白质的内切核酸酶。它不处理RNA酶A样活性。该酶能够在体外切割c-myc CRD RNA以外的RNA。它不依赖于Mg 2+，对EDTA有抗性。核酸内切酶在70 ℃及以上失活。这些特性将该酶与先前描述的其他脊椎动物核酸内切酶区分开来。
Endonuclease-mediated mRNA decay appears to be a common mode of mRNA degradation in mammalian cells, but yet only 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 similar to 10-35kDa size copurified 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 is 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'hydroxly group, and it appears to be protein-only endonuclease. It does not process RNase A-like activity. The enzyme is capable of cleaving RNAs other than c-myc CRD RNA in vitro. It is Mg2+-independant 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.