Piller K J, Rusché L N, Cruz-Reyes J, Sollner-Webb B
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
RNA. 1997 Mar;3(3):279-90.
RNA editing in kinetoplastids, the specific insertion and deletion of U residues, requires endonuclease cleavage of the pre-mRNA at each cycle of insertion/deletion. We have resolved three endoribonuclease activities from Trypanosoma brucei mitochondrial extracts that cleave CYb pre-mRNA specifically. One of these, which sediments at approximately 20S and is not affected substantially by DTT, has all the features of the editing endonuclease. It cleaves CYb pre-edited or partially edited mRNA only when annealed to the anchor region of a cognate guide RNA (gRNA), and it cleaves accurately just 5' of the duplex region. Its specificity is for the 5' end of extended duplex RNA regions, and this prevents cleavage of the gRNA or other positions in the mRNA. This gRNA-directed nuclease is evidently the same activity that functions in A6 pre-mRNA editing. However, it is distinct and separable from a previously observed DTT-requiring endonuclease that sediments similarly under certain conditions, but does not cleave precisely at the first editing site in either the presence or absence of a gRNA. The editing nuclease is also distinct from a DTT-inhibited endonuclease that cleaves numerous free pre-mRNAs at a common structure in the region of the first editing site.
动质体中的RNA编辑，即U残基的特异性插入和缺失，在每次插入/缺失循环中都需要对前体mRNA进行核酸内切酶切割。我们从布氏锥虫线粒体提取物中解析出了三种特异性切割CYb前体mRNA的核糖核酸内切酶活性。其中一种在约20S处沉降且基本不受二硫苏糖醇（DTT）影响，具有编辑核酸内切酶的所有特征。它仅在与同源引导RNA（gRNA）的锚定区域退火时才切割CYb预编辑或部分编辑的mRNA，并且在双链区域的5'端精确切割。其特异性针对延伸双链RNA区域的5'端，这可防止切割gRNA或mRNA中的其他位置。这种gRNA导向的核酸酶显然与在A6前体mRNA编辑中起作用的活性相同。然而，它与先前观察到的在某些条件下类似沉降但无论有无gRNA都不在第一个编辑位点精确切割的需要DTT的核酸内切酶不同且可分离。编辑核酸酶也不同于一种受DTT抑制的核酸内切酶，后者在第一个编辑位点区域的共同结构处切割众多游离的前体mRNA。
