Aphasizhev Ruslan, Aphasizheva Inna, Simpson Larry
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10617-22. doi: 10.1073/pnas.1833120100. Epub 2003 Sep 3.
The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3' terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3' oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purification-tagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3' end of a single-stranded RNA, thereby confirming that RET2 is a 3' TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro U-insertion editing and has no effect on the length of the 3' oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3' end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.
锥虫线粒体中mRNA特定位点U残基的插入和缺失被认为涉及3'末端尿苷酰转移酶（TUTase）活性。产生反式作用向导RNA（gRNA）的非编码3'寡聚[U]尾也需要TUTase活性。我们已经描述了两种TUTase，RET1（RNA编辑TUTase 1）和RET2（RNA编辑TUTase 2），它们是不同编辑复合体的组成部分。通过去除线粒体信号序列，表达了串联亲和纯化标签的布氏锥虫RET2（TbRET2），并将其定位于热带利什曼原虫细胞的细胞质中。双重亲和分离得到了带有标签的TbRET2以及一些其他蛋白质。这种物质表现出一种U特异性转移酶活性，其中单个U被添加到单链RNA的3'末端，从而证实RET2是一种3' TUTase。我们还发现，在布氏锥虫中对RET2表达进行RNA干扰会抑制体外U插入编辑，并且对gRNA的3'寡聚[U]尾的长度没有影响，而RET1的下调对体外U插入编辑有轻微影响，但会导致寡聚[U]尾的平均长度减少。这一发现表明，RET2负责编辑位点的U插入，而RET1参与gRNA 3'末端成熟，这对于产生功能性gRNA至关重要。根据这些结果，我们将先前描述的包含RET1的TUT-II复合体在功能上重新标记为向导RNA加工复合体。
