TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites
TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites
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DOI：
10.1261/rna.2285510
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发表时间：
2010-11-01
期刊：
影响因子：
4.5
通讯作者：
Read, Laurie K.
中科院分区：
文献类型：
--
作者：
Ammerman, Michelle L.;Presnyak, Vladimir;Read, Laurie K.
TbRGG2 是一种重要的动质体 RNA 编辑辅助因子，专门作用于泛编辑 RNA。为了了解 TbRGG2 的作用机制，我们对 TbRGG2 敲低细胞中编辑的 RNA 群体进行了深入分析，并对蛋白质的生化活性进行了体外检查。我们证明，TbRGG2 下调对泛编辑 RNA 5' 端的编辑影响比对 3' 端的影响更严重。在 TbRGG2 敲低细胞中，编辑的启动在一定程度上减少。此外，TbRGG2 发挥启动后作用，因为在 TbRGG2 耗尽的细胞中编辑变得停滞，导致编辑的 3' 到 5' 进程总体减少。对来自野生型和 TbRGG2 耗尽细胞的编辑 RNA 的详细分析表明，TbRGG2 促进编辑过去的内在暂停位点的进展，这些位点通常对应于同源引导 RNA (gRNA) 的 3' 末端。此外，在 TbRGG2 耗尽的细胞中，非规范编辑的连接区域要么不存在，要么显着缩短，这与受损的 gRNA 转换一致。序列分析进一步表明 TbRGG2 促进某些 gRNA 的完全利用。体外 RNA 退火和体内 RNA 解旋测定表明 TbRGG2 可以调节 RNA-RNA 相互作用。总的来说，这些数据与 TbRGG2 通过其影响 gRNA 利用的能力（在特定 gRNA 之间的转换期间和某些 gRNA 使用期间）促进编辑的启动和 3' 至 5' 进展的模型一致。
TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wildtype and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.