Revisiting replicating RNAs DNA-dependent RNA polymerases are well known for their ability to produce RNA from DNA templates. Much less is known about their noncanonical activity: the generation and replication of RNA from RNA templates. Deeper insights into this process could shed light on questions relating to the origin of life, molecular evolution, and the replication of certain RNA pathogens such as hepatitis delta virus and plant viroids. Jain et al. explore in detail how the bacteriophage T7 RNA polymerase generates and amplifies diverse RNA sequences in vitro. Using sequencing, microfluidics, and bioinformatics, they chart the emergence and evolution of replicating RNA motifs and identify mechanisms that explain their selection and structures. Science, this issue p. eaay0688 A detailed study of de novo RNA generation by a DNA-dependent RNA polymerase sheds light on the origin of RNA replicons. INTRODUCTION Although genetic information is commonly encoded in DNA and transmitted by means of DNA-templated DNA replication, RNA can also be transferred as hereditary material through RNA-templated RNA replication. Two classes of protein-catalyzed RNA replication systems have been described. In the first, specialized RNA-dependent RNA polymerases replicate the genomes of RNA viruses such as influenza and dengue. In the second, cellular enzymes normally involved in the transcription of DNA to RNA can copy certain RNAs such as plant viroids and human hepatitis delta virus. The diversity of RNAs using DNA-dependent RNA polymerases for replication and the underlying molecular mechanisms have not been fully explored. RATIONALE Five RNA sequences that can be replicated in vitro by bacteriophage T7 DNA-dependent RNA polymerase (T7 RNAP) have previously been described. The origins of these replicating RNAs and requirements for replication by T7 RNAP are unclear. We applied next-generation sequencing, microfluidics, and bioinformatics to address (i) how a DNA-dependent RNA polymerase can replicate RNA, (ii) which RNA templates are efficiently replicated, and (iii) how replicating RNAs originate. RESULTS We set up a series of T7 RNAP reactions with no explicitly added templates. These reactions yielded RNA replicons with different sequences but a consistent structural framework defined by a two-way repeat (a long inverted repeat throughout the RNA length) and a four-way repeat (a shorter inverted repeat embedded within each arm of the two-way repeat). We showed that two-way and four-way repeats are required for efficient RNA replication by T7 RNAP, suggesting that RNAs with two-way and four-way repeats arise in no-template-added reactions by “survival of the fittest” in the test tube. The requirement of two-way repeats further implies that RNAs form a long hairpin structure, whereas the four-way–repeat requirement suggests that RNAs change structure during replication—what we refer to as RNA shape-shifting. From experiments with chemically synthesized RNA templates, we also identified a nontemplating 3′ nucleotide extension as a critical requirement for the initiation of replication. This requirement suggests that initiation of new RNA products occurs internally within the template sequence, which we call subterminal de novo initiation. We additionally found that RNA templates with defined 5′ and 3′ ends can be used processively for multiple rounds of RNA synthesis. Such a mechanism, which we term interrupted rolling-circle synthesis, yields RNA products consisting of multiple repeats of template sequence. RNAs synthesized in no-template-added T7 RNAP reactions could have been replication products of preexisting sequences or products of a de novo process. To distinguish between these two possibilities, we isolated hundreds of RNA replicons by scaling up our experimental throughput using microfluidics. Analysis of our large replicon repertoire led us to hypothesize that replicating RNAs can originate de novo through partial instruction from DNA seeds. In support of this hypothesis, we demonstrated the formation of novel replicating RNAs from a DNA seed pool of our own choosing. We observed that RNA replicons consisted of DNA seed-sequence information that had been duplicated and reduplicated to yield the characteristic four-way–repeat pattern, suggesting a specific cascade of steps of polymerase activity and molecular evolution leading to the origin of replicating RNAs. CONCLUSION Our results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could serve as hereditary material in cellular contexts. Origins of RNAs replicated by a DNA-dependent RNA polymerase (T7 RNAP). (Top) A diversity of RNA replicons was obtained from no-template-added T7 RNAP reactions set up as aqueous droplets in oil using microfluidics. Replicons consistently exhibited a structural framework of two-way repeats (blue arrows) and four-way repeats (orange arrows). NTPs, nucleoside triphosphates. (Middle) Replicons with paired two-way and paired four-way repeats amplified more efficiently and outcompeted other RNAs (gray boxes, paired nucleotides; white boxes, unpaired nucleotides). (Bottom) Novel replicons can originate from DNA seeds through DNA-templated transcription, two-way– and four-way–repeat generation, and Darwinian selection. The green boxes on the right show the input seed sequences. Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.

中文翻译：

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转录聚合酶催化新型 RNA 复制子的出现


重新审视复制 RNA DNA 依赖性 RNA 聚合酶以其从 DNA 模板产生 RNA 的能力而闻名。人们对它们的非常规活动（从 RNA 模板生成和复制 RNA）知之甚少。更深入地了解这一过程可以揭示与生命起源、分子进化以及某些 RNA 病原体（如丁型肝炎病毒和植物类病毒）的复制相关的问题。贾恩等人。详细探索噬菌体 T7 RNA 聚合酶如何在体外生成和扩增不同的 RNA 序列。他们利用测序、微流体和生物信息学，绘制了复制 RNA 基序的出现和进化图，并确定了解释其选择和结构的机制。科学，本期第 14 页。 eaay0688 对 DNA 依赖性 RNA 聚合酶从头生成 RNA 的详细研究揭示了 RNA 复制子的起源。简介 虽然遗传信息通常在 DNA 中编码并通过以 DNA 为模板的 DNA 复制来传递，但 RNA 也可以通过以 RNA 为模板的 RNA 复制作为遗传物质进行传递。已经描述了两类蛋白质催化的 RNA 复制系统。首先，专门的RNA依赖性RNA聚合酶复制流感病毒和登革热等RNA病毒的基因组。在第二种情况下，通常参与 DNA 转录为 RNA 的细胞酶可以复制某些 RNA，例如植物类病毒和人类丁型肝炎病毒。使用 DNA 依赖性 RNA 聚合酶进行复制的 RNA 的多样性及其潜在的分子机制尚未得到充分探索。基本原理 先前已经描述了可通过噬菌体 T7 DNA 依赖性 RNA 聚合酶 (T7 RNAP) 在体外复制的 5 种 RNA 序列。 这些复制 RNA 的起源以及 T7 RNAP 复制的要求尚不清楚。我们应用下一代测序、微流体和生物信息学来解决（i）DNA依赖性RNA聚合酶如何复制RNA，（ii）哪些RNA模板可以有效复制，以及（iii）复制RNA是如何起源的。结果我们建立了一系列 T7 RNAP 反应，没有明确添加模板。这些反应产生了具有不同序列的RNA复制子，但具有一致的结构框架，由双向重复（整个RNA长度上的长反向重复）和四向重复（嵌入双向重复的每个臂中的较短反向重复）定义。重复）。我们表明，T7 RNAP 的有效 RNA 复制需要双向和四向重复，这表明在不添加模板的反应中，通过“适者生存”，具有双向和四向重复的 RNA 会出现。试管。双向重复的要求进一步意味着RNA形成长发夹结构，而四向重复的要求表明RNA在复制过程中改变结构——我们称之为RNA形状转变。通过化学合成 RNA 模板的实验，我们还发现非模板 3' 核苷酸延伸是复制起始的关键要求。这一要求表明新 RNA 产物的启动发生在模板序列内部，我们称之为亚末端从头启动。我们还发现，具有明确 5' 和 3' 末端的 RNA 模板可以连续用于多轮 RNA 合成。这种机制，我们称之为间断滚环合成，产生由模板序列的多次重复组成的RNA产物。 在不添加模板的 T7 RNAP 反应中合成的 RNA 可能是预先存在的序列的复制产物或从头过程的产物。为了区分这两种可能性，我们通过使用微流体扩大实验通量，分离了数百个 RNA 复制子。对我们庞大的复制子库的分析使我们假设复制 RNA 可以通过 DNA 种子的部分指令从头开始。为了支持这一假设，我们证明了从我们自己选择的 DNA 种子库中形成新型复制 RNA。我们观察到RNA复制子由DNA种子序列信息组成，这些信息经过一再复制以产生特征性的四向重复模式，这表明聚合酶活性和分子进化步骤的特定级联导致了复制RNA的起源。结论 我们的结果为自然发生的 RNA 遗传元件的起源和复制模型提供了信息，并提出了一种方法，使不同的 RNA 群体可以作为细胞环境中的遗传材料。由 DNA 依赖性 RNA 聚合酶 (T7 RNAP) 复制的 RNA 的起源。 （上）使用微流体从不添加模板的 T7 RNAP 反应中获得了多种 RNA 复制子，该反应设置为油中的水滴。复制子始终表现出双向重复（蓝色箭头）和四向重复（橙色箭头）的结构框架。 NTP，三磷酸核苷。 （中）具有配对双向和配对四向重复序列的复制子可以更有效地扩增，并在竞争中胜过其他 RNA（灰色框，配对核苷酸；白色框，未配对核苷酸）。 （下）新型复制子可以通过 DNA 模板转录、双向和四向重复生成以及达尔文选择源自 DNA 种子。右侧的绿色框显示输入种子序列。转录聚合酶可以表现出一种从 RNA 再生某些 RNA 模板的不寻常模式，产生可以以 RNA 作为信息载体复制和进化的系统。两类致病性 RNA（动物中的丁型肝炎病毒和植物中的类病毒）由宿主转录聚合酶复制。使用 T7 噬菌体转录聚合酶进行的体外 RNA 复制作为实验模型，我们鉴定了数百种新的复制 RNA，定义了复制的三个机制标志（亚末端从头起始、RNA 变形和中断的滚环合成），并描述了 DNA 种子的出现作为新型 RNA 复制子起源的机制。这些结果为自然发生的 RNA 遗传元件的起源和复制模型提供了信息，并提出了一种方法，通过该方法可以使不同的 RNA 群体作为遗传物质在细胞环境中繁殖。