Tafech Alaeddin, Bennett William R, Mills Fergil, Lee Chow H
Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, Canada BC V2N 4Z9.
Biochim Biophys Acta. 2007 Jan;1769(1):49-60. doi: 10.1016/j.bbaexp.2006.11.009. Epub 2006 Dec 15.
The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705-1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705-1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.
包含编码区决定簇（CRD）核苷酸（nts）1705 - 1792的c-myc mRNA编码区在调节c-myc mRNA稳定性方面至关重要。这部分是由于c-myc CRD RNA易受核糖核酸内切酶攻击。我们之前已纯化并鉴定了一种在体外切割c-myc CRD RNA的哺乳动物核糖核酸内切酶。该酶初步被鉴定为一种35 kDa的RNase1样核糖核酸内切酶。为了了解该酶切割RNA的序列和二级结构要求，我们使用核糖核酸酶探测技术确定了c-myc CRD RNA nts 1705 - 1792的二级结构。c-myc CRD RNA的二级结构有五个茎；其中两个含有4个碱基对（茎I和V），三个由3个碱基对组成（茎II、III和IV）。使用c-myc CRD和几个c-myc CRD突变体作为底物进行的内切核酸酶分析得出以下结论：（i）该酶倾向于在单链区域的二核苷酸UA、CA和UG之间切割；（ii）该酶对UA二核苷酸更具特异性。这些特性进一步将该酶与之前描述的在体外切割c-myc mRNA的哺乳动物内切酶区分开来。
