Osato Daren, Rogers Kestrel, Guo Qiang, Li Feng, Richmond Greg, Klug Felix, Simpson Larry
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA.
RNA. 2009 Jul;15(7):1338-44. doi: 10.1261/rna.1642809. Epub 2009 May 15.
The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a approximately 1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (La) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (Lb) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The Lb-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and Lb-complex gradient fractions were similar in composition and Lb-complex bands often degraded to L-complex bands after manipulation or freeze-thaw cycles. The La-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The Lb-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the Lb-complexes. The MRP1/2 RNA binding complex is localized mainly in the Lb-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the Lb-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or "editosome."
含RNA连接酶的复合物或L复合物是锥虫线粒体中参与尿苷插入/缺失RNA编辑的核心复合物。用TAP标签标记的编辑蛋白LC-8（TbMP44/KREPB5）转染的细胞线粒体裂解物经甘油梯度分离后的蓝色非变性凝胶显示，有一条约1 MDa的L复合物条带，此外，还有两个分子量稍大的含REL1的较小复合物：一个（La）与L复合物共沉降，在凝胶中迁移至约1.2 MDa处；另一个（Lb）显示分子量从1 MDa持续增加至沉降超过70S的颗粒。Lb复合物似乎主要由L复合物成分组成，因为L复合物和Lb复合物梯度级分的多肽谱在组成上相似，并且Lb复合物条带在操作或冻融循环后常降解为L复合物条带。La复合物可能是人为造成的，因为这种凝胶迁移可由各种实验操作产生。然而，这种变化的性质和任何细胞作用仍有待确定。来自裂解物和TAP下拉实验的Lb复合物对核糖核酸酶A消化敏感，表明RNA与Lb复合物的稳定性有关。MRP1/2 RNA结合复合物主要以亚化学计量的量定位于Lb复合物中，并且这种结合对核糖核酸酶敏感。我们认为，Lb复合物可能提供一个支架，在编辑过程中与其他编辑因子进行动态相互作用，以形成活性全酶或“编辑体”。
