Law Julie A, O'Hearn Sean, Sollner-Webb Barbara
Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Mol Cell Biol. 2007 Jan;27(2):777-87. doi: 10.1128/MCB.01460-06. Epub 2006 Nov 13.
In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an approximately 20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the approximately 20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as approximately 10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the approximately 20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.
在锥虫RNA编辑过程中，尿苷酸（U）残基通过一个约20S的蛋白质复合物在线粒体前体mRNA的多个位点插入和缺失，该复合物催化切割、添加U/去除U以及连接的循环。我们利用RNA干扰耗尽了TbMP18（第七条带），这是我们纯化的编辑复合物中最后一个未经检测的主要蛋白质，结果表明它是必不可少的。TbMP18对于U缺失和U插入切割以及约20S编辑复合物的完整性至关重要，该复合物的其他主要成分，即TbMP99、TbMP81、TbMP63、TbMP52、TbMP48、TbMP42（第一条至第六条带）和TbMP57，反而以约10S的缔合形式沉降。此外，TbMP18增强了TbMP57末端U转移酶对编辑底物的识别，可能有助于识别成分TbMP81。在没有TbMP18的情况下，其他编辑活性及其在预切割编辑中的协调作用仍然活跃。这些数据让人想起A. Schnaufer等人（《分子细胞》12:307 - 319，2003年）报道的关于编辑亚复合物的数据，并表明这些亚复合物如先前所提出的那样通过TbMP18在约20S复合物中聚集在一起。我们的数据还暗示，这些蛋白质在这些亚复合物中的寿命比在完整编辑复合物中的寿命短。当编辑复合物碎片化时，如在TbMP18耗尽时，编辑内切核酸酶切割的丧失对锥虫应该是有利的，可将断裂的mRNA数量降至最低。
