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

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 Ψ. 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 Ψ 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.目前已在生命之树（tree of life）各分支的RNA上发现超过170种修饰类型。长期以来，系统解析RNA修饰的主要瓶颈在于缺乏能够规模化、系统性获取多种RNA修饰高可信度图谱的研究方法。本研究开发了Pan-Mod-seq技术，可实现数十个样本中16种不同RNA修饰的并行、灵敏且特异性的从头（de novo）鉴定。我们将Pan-Mod-seq应用于覆盖所有3个生命域的14个不同物种的RNA样本，其中多数样本取自化学、物理及生物学条件高度可变的梯度环境，旨在系统性探究核糖体RNA（ribosomal RNA, rRNA）修饰的可塑性。尽管在嗜温微生物中，动态修饰位点相对罕见，但我们发现这类位点在超嗜热微生物中广泛存在，其中约50%已鉴定的修饰会随温度变化被诱导产生。我们重点解析了3种关键的动态诱导型修饰：5-甲基胞嘧啶（m5C）、N4-乙酰胞嘧啶（ac4C）与假尿苷（pseudouridine, Ψ）。我们发现了一套覆盖两个核糖体RNA亚基数十个靶位点的m5C修饰调控程序，在两个被采样的超嗜热微生物中，所有这些靶位点的修饰均会随生长温度升高被显著诱导。在这两个物种中，m5C修饰均通过单一酶在单一共有基序处完成，而该酶对于微生物在高温下的生长至关重要。与之类似，在所有3个被采样的超嗜热微生物中，ac4C修饰均会在数百个位点处随温度动态诱导产生，且同样识别单一共有基序。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。两种修饰酶的温度依赖性诱导均可在体外重现，这证明该调控过程是两种酶的内在特性，而非由外部因素调控。我们获取了野生型（wild type, WT）菌株在高低温条件下，以及甲基化酶缺失菌株的核糖体高分辨率冷冻电镜（cryogenic 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，我们还发现柯达热球菌（Thermococcus kodakarensis）中Ψ修饰水平会随温度系统性升高，并揭示该过程主要由单一温度诱导的小RNA（small RNA, sRNA）介导：该小RNA可指导前所未有的大量靶位点发生假尿苷化，其缺失会导致微生物在高温下生长缺陷。本研究的发现为生命之树各分支代表性物种的rRNA修饰可塑性提供了全面且系统性的视角，解析了不同修饰在高温条件下对核糖体稳定性的贡献，并开发了一种可前所未有地解析健康与疾病状态下rRNA表观转录组的研究方法。