Leavitt Jesse S, Moore Henry, Santangelo Thomas J, Lowe Todd M
Department of Biomolecular Engineering, Baskin School of Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, 80523, USA.
bioRxiv. 2025 May 5:2025.05.02.651894. doi: 10.1101/2025.05.02.651894.
Transfer RNA (tRNA) modifications play essential roles in structure, stability and decoding accuracy, yet the evolutionary dynamics and diversity of these modifications remain incompletely understood. Recent advances in high-throughput sequencing, including Ordered Two-Template Relay sequencing (OTTR-seq), now facilitate systematic, transcriptome-wide detection of tRNA modifications at single-base resolution. Here we employed OTTR-seq to comprehensively profile tRNA modifications across nine archaeal species spanning diverse ecological niches, including thermophiles, methanogens, acidophiles, and halophiles. We identified coordinated and mutually exclusive methylation patterns at acceptor stem position 6 and 67 in hyperthermophiles, as well as unexpected clade-specific co-modifications at core positions 10 and 26, demonstrating tolerance of canonical anti-determinants in Thermoprotei. Comparative genomic analyses revealed clear associations between modifications and evolutionary changes in enzyme domain architectures, including divergence in homologs of Trm14, Trm10, Trm11, and Trm1. We also expanded upon previously characterized identity elements in the D-stem, such as the conserved G10oU25 pair critical for Trm11 specificity, providing additional insight into determinants that likely govern enzyme-substrate interactions. Together, these findings offer valuable insights into archaeal tRNA modification biology and evolutionary dynamics, and provide a foundation for guiding targeted biochemical experiments and mechanistic studies.
转运RNA（tRNA）修饰在结构、稳定性和解码准确性方面发挥着重要作用，然而这些修饰的进化动态和多样性仍未得到充分理解。高通量测序技术的最新进展，包括有序双模板中继测序（OTTR-seq），现在有助于在单碱基分辨率下对tRNA修饰进行系统的全转录组检测。在这里，我们使用OTTR-seq对跨越不同生态位的九种古菌物种的tRNA修饰进行了全面分析，这些古菌包括嗜热菌、产甲烷菌、嗜酸菌和嗜盐菌。我们在嗜热超嗜热菌的受体茎位置6和67处鉴定出了协同和相互排斥的甲基化模式，以及在核心位置10和26处意外的分支特异性共修饰，这表明嗜热栖热菌中对典型反决定簇具有耐受性。比较基因组分析揭示了修饰与酶结构域结构的进化变化之间的明确关联，包括Trm14、Trm10、Trm11和Trm1同源物的差异。我们还扩展了先前在D茎中表征的识别元件，例如对Trm11特异性至关重要的保守G10oU25对，并进一步深入了解了可能控制酶-底物相互作用的决定因素。总之，这些发现为古菌tRNA修饰生物学和进化动态提供了有价值的见解，并为指导有针对性的生化实验和机制研究奠定了基础。
