Igo R P, Palazzo S S, Burgess M L, Panigrahi A K, Stuart K
Seattle Biomedical Research Institute, Seattle, Washington 98109, USA.
Mol Cell Biol. 2000 Nov;20(22):8447-57. doi: 10.1128/MCB.20.22.8447-8457.2000.
RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5' fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5' fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.
布氏锥虫中的RNA编辑在线粒体前体mRNA中，在引导RNA（gRNA）的指导下插入和删除尿苷酸（U）。我们在此报告一种新型体外预切割编辑分析方法的开发及其用于研究插入RNA编辑中U添加和RNA连接步骤的gRNA特异性。底物RNA的5'片段随着gRNA指定添加的U的数量而积累，并且具有超过指定数量U的U添加产物很少见。在没有连接的情况下发生了达到指定数量的U添加，但U添加产物的积累减慢。具有正确数量添加U的5'片段优先被连接，显然是由腺苷酸化的RNA连接酶连接，因为不需要外源添加ATP，并且由于用焦磷酸处理消除了连接。当编辑位点上游的前体mRNA是单链时，在没有连接的情况下gRNA指定的U添加是明显的，当它与gRNA碱基配对时更是如此。这些结果表明，U添加和RNA连接步骤都有助于RNA编辑的精确性。
