Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata2

Over 170 modifications are deposited on RNA, across the tree of life. A major bottleneck to systematically dissecting RNA modifications has been the lack of methods allowing to acquire systematic, high-confidence maps of many RNA modifications and at scale. Here we have developed Pan-Mod-seq, which permits de-novo, sensitive and specific identification of 16 distinct modifications in dozens of samples in parallel. We applied Pan-Mod-seq to RNA from 14 different species from all 3 domains of life, most of which sampled under highly variable chemical/physical/biological gradients, aiming to systematically explore the plasticity of rRNA modifications. While dynamically modified sites are relatively rare in mesophiles, we find that these are widespread in hyperthermophiles, where ~50% of identified modifications were induced with temperature. We focused on dissection of three key dynamically induced modifications: m5C, ac4C and ?. We uncover an m5C program encompassing dozens of targets across both rRNA subunits, all of which are dramatically induced with growth temperature in two of the sampled hyperthermophiles. In both cases m5C is introduced at a single consensus motif, via a single enzyme which is essential for growth at higher temperatures. ac4C in turn is dynamically induced among all three sampled hyperthermophiles at hundreds of sites, also at a single consensus motif. Remarkably, the methylation and acetylation overlap, and together form a tandemly modified temperature induced G-m5C-ac4C-G sequence motif. Temperature-dependent induction of both enzymes can be recapitulated in vitro, establishing that it is an intrinsic property of the two enzymes rather than externally regulated. We obtain high-resolution cryoEM structures of ribosomes from WT strains at low and high temperatures and ones deleted of the methylating enzyme, and identify numerous mechanisms via which m5C confers structural stability. Finally, we also uncover a systematic induction in ? levels in T. kodakarensis, and reveal that it is largely coordinated by a single temperature-induced sRNA guiding pseudouridine formation at an unprecedented number of targets, whose loss results in growth deficiency at higher temperatures. Our findings offer a wide and systematic view of rRNA modification plasticity across representative species sampled from the tree of life, dissect the ability of different modifications to contribute to the stability of ribosomes at higher temperatures, and present a methodology allowing an unprecedented view on the the rRNA epitranscriptome in health and in disease. Overall design: In-vitro methylation followed by bisulfite sequencing. A mixture of total RNA of Saccharomyces cerevisiae (WT) and Pyrococcus furiosus (?RsmB) were in-vitro methylated with either Pfu-RsmB or TK1935under different temperatures (37 °C, 55 °C, 75 °C, 85 °C, 95 °C, and 105 °C), and a no enzyme control kept at 4 °C. Samples were subjected to bisulfite sequencing. One replicate per condition.

在整个生命之树的各类生物中，RNA上已报道存在超过170种修饰。长期以来，系统解析RNA修饰的主要瓶颈在于缺乏能够大规模、系统性获取多种RNA修饰高精度定位图谱的技术手段。本研究开发了Pan-Mod-seq技术，可实现数十个样本并行的、高灵敏度与特异性的从头鉴定16种不同RNA修饰。

我们将Pan-Mod-seq应用于来自生命三大域的14个不同物种的RNA样本，其中多数样本取自化学、物理、生物条件高度多变的环境，旨在系统性探究核糖体RNA（ribosomal RNA, rRNA）修饰的可塑性。尽管动态修饰位点在嗜温生物中较为罕见，但我们发现这类位点在超嗜热生物中广泛存在，其中约50%的已鉴定修饰可被温度诱导。

本研究聚焦解析三种关键的温度诱导动态修饰：m5C、ac4C以及假尿苷（pseudouridine, Ψ）。我们发现了一套m5C修饰调控网络，该网络覆盖两个核糖体RNA亚基的数十个靶标位点，在两个被采样的超嗜热生物中，所有这些位点的修饰水平均随生长温度显著上调。在这两个物种中，m5C修饰均通过单一酶在单一保守基序处完成添加，且该酶对生物在高温下的生长至关重要。而ac4C修饰则在所有三个被采样的超嗜热生物的数百个位点上呈现温度动态诱导特征，同样仅识别单一保守基序。值得注意的是，甲基化与乙酰化修饰存在位点重叠，二者共同构成了温度诱导的串联修饰序列基序G-m5C-ac4C-G。

两种酶的温度依赖性诱导均可在体外重现，这表明该调控过程是两种酶的固有属性，而非受外部因素调控。我们解析了不同温度下野生型（Wild Type, WT）菌株以及甲基化酶敲除菌株的核糖体高分辨率冷冻电镜（cryo-electron microscopy, cryoEM）结构，并阐明了m5C修饰赋予核糖体结构稳定性的多种机制。最后，我们还在柯达热球菌（Thermococcus kodakarensis, T. kodakarensis）中发现了假尿苷水平的系统性上调，并揭示该过程主要由单一温度诱导的小RNA（small RNA, sRNA）介导完成：该sRNA可指导前所未有的大量靶标位点发生假尿苷修饰，其缺失会导致生物在高温下生长缺陷。

本研究的发现为来自生命之树各分支的代表性物种的rRNA修饰可塑性提供了全面且系统性的视角，解析了不同RNA修饰在高温下对核糖体稳定性的贡献机制，并展示了一种可前所未有地解析健康与疾病状态下rRNA表观转录组（epitranscriptome）的技术方法。

实验整体设计：先进行体外甲基化反应，随后开展亚硫酸氢盐测序。将酿酒酵母（Saccharomyces cerevisiae，WT）与激烈火球菌（Pyrococcus furiosus，ΔRsmB）的总RNA混合，分别使用Pfu-RsmB或TK1935在不同温度（37℃、55℃、75℃、85℃、95℃、105℃）下进行体外甲基化，同时设置无酶对照组并置于4℃环境。所有样本均进行亚硫酸氢盐测序，每个条件设置一次生物学重复。