Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA Editing Substrate binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C). RESC and REH2C stably co-purify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include mis-edited 'junction' regions that match neither pre-mRNA nor fully-edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine Ribosomal Protein Subunit 12 (RPS12) and ATPase-subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5' positions but increased total editing at examined A6 3' positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5' nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3'-terminal adenines in gRNA-1 could direct a non-canonical 2U-insertion that causes major pausing in 3'-5' progression. In A6, a non-canonical sequence element that depends on REH2C in a region normally targeted by the 3'-half of gRNA-1 may hinder early-editing progression. Overall, we defined transcript-specific effects of REH2C loss.

中文翻译：

---

锥虫中解旋酶复合物对精确 mRNA 编辑的位点特异性和 mRNA 特异性控制

线粒体 mRNA 中的锥虫 U 插入/删除 RNA 编辑涉及引导 RNA (gRNA) 和辅助 RNA 编辑底物结合复合物 (RESC) 和 RNA 编辑解旋酶 2 复合物 (REH2C)。RESC 和 REH2C 与编辑 mRNA 稳定地共纯化，但这些复合物之间的功能相互作用仍不清楚。大多数稳态 mRNA 都经过部分编辑，并包含错误编辑的“连接”区域，这些区域既不匹配前 mRNA，也不匹配完全编辑的转录本。编辑特异性对于线粒体 RNA 成熟和功能至关重要，但其基本控制机制仍不清楚。在这里，我们应用了一种新的核苷酸分辨率 RNA-seq 方法来检查核糖体蛋白亚基 12 (RPS12) 和 ATPase 亚基 6 (A6) mRNA 转录本。我们直接将 RESC 和 REH2C 相关转录本与线粒体总 RNA 中发现的转录本进行比较。RESC 相关转录本在全面且准确的编辑中表现出位点优先富集。REH2C 功能丧失在总 RNA 和 RESC 相关 RNA 中诱导类似的底物特异性和位点特异性编辑效应。它主要减少了 RPS12 5' 位置的总编辑，但增加了检查的 A6 3' 位置的总编辑。REH2C 功能丧失导致两个转录本中的精确编辑位点优先丧失。然而，总编辑量或准确编辑量的变化并不一定涉及共同站点。起始 gRNA (gRNA-1) 的几个 5' 核苷酸指导两个转录本中的精确编辑。然而，在 RPS12 中，gRNA-1 中的两个保守的 3' 末端腺嘌呤可以引导非规范的 2U 插入，从而导致 3'-5' 进展的主要暂停。在 A6 中，依赖于通常由 gRNA-1 3'-半部分靶向的区域中的 REH2C 的非规范序列元件可能会阻碍早期编辑进程。总的来说，我们定义了 REH2C 损失的转录特异性影响。