Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
ACS Chem Biol. 2024 Feb 16;19(2):348-356. doi: 10.1021/acschembio.3c00555. Epub 2024 Jan 22.
A-to-I editing catalyzed by adenosine deaminase acting on RNAs impacts numerous physiological and biochemical processes that are essential for cellular functions and is a big contributor to the infectivity of certain RNA viruses. The outcome of this deamination leads to changes in the eukaryotic transcriptome functionally resembling A-G transitions since inosine preferentially pairs with cytosine. Moreover, hyper-editing or multiple A to G transitions in clusters were detected in measles virus. Inosine modifications either directly on viral RNA or on cellular RNA can have antiviral or pro-viral repercussions. While many of the significant roles of inosine in cellular RNAs are well understood, the effects of hyper-editing of A to I on viral polymerase activity during RNA replication remain elusive. Moreover, biological strategies such as molecular cloning and RNA-seq for transcriptomic interrogation rely on RT-polymerase chain reaction with little to no emphasis placed on the first step, reverse transcription, which may reshape the sequencing results when hypermodification is present. In this study, we systematically explore the influence of inosine modification, varying the number and position of inosines, on decoding outcomes using three different reverse transcriptases (RTs) followed by standard Sanger sequencing. We find that inosine alone or in clusters can differentially affect the RT activity. To gain structural insights into the accommodation of inosine in the polymerase site of HIV-1 reverse transcriptase (HIV-1-RT) and how this structural context affects the base pairing rules for inosine, we performed molecular dynamics simulations of the HIV-1-RT. The simulations highlight the importance of the protein-nucleotide interaction as a critical factor in deciphering the base pairing behavior of inosine clusters. This effort sets the groundwork for decrypting the physiological significance of inosine and linking the fidelity of reverse transcriptase and the possible diverse transcription outcomes of cellular RNAs and/or viral RNAs where hyper-edited inosines are present in the transcripts.
腺苷脱氨酶作用于 RNA 的 A 到 I 编辑催化对许多生理和生化过程产生影响，这些过程对细胞功能至关重要，是某些 RNA 病毒感染力的主要贡献者。这种脱氨作用的结果导致真核转录组发生变化，其功能类似于 A-G 转换，因为肌苷优先与胞嘧啶配对。此外，在麻疹病毒中检测到高编辑或簇中的多个 A 到 G 转换。病毒 RNA 或细胞 RNA 上的肌苷修饰可能具有抗病毒或促病毒作用。虽然肌苷在细胞 RNA 中的许多重要作用已经得到很好的理解，但在 RNA 复制过程中 A 到 I 的高编辑对病毒聚合酶活性的影响仍然难以捉摸。此外，用于转录组研究的分子克隆和 RNA-seq 等生物学策略依赖于 RT-聚合酶链反应，几乎没有强调第一步，即反转录，当存在高度修饰时，反转录可能会改变测序结果。在这项研究中，我们系统地研究了肌苷修饰（改变肌苷的数量和位置）对使用三种不同逆转录酶 (RT) 进行解码结果的影响，然后进行标准 Sanger 测序。我们发现，肌苷单独或成簇存在会对 RT 活性产生不同的影响。为了深入了解肌苷在 HIV-1 逆转录酶 (HIV-1-RT) 聚合酶位点中的结构适应性，以及这种结构背景如何影响肌苷的碱基配对规则，我们对 HIV-1-RT 进行了分子动力学模拟。模拟结果强调了蛋白质-核苷酸相互作用作为破译肌苷簇碱基配对行为的关键因素的重要性。这项工作为破译肌苷的生理意义奠定了基础，并将逆转录酶的保真度与存在高度编辑肌苷的细胞 RNA 和/或病毒 RNA 的转录结果多样性联系起来。
