古菌RNA加工流转关键酶生理生化功能及分子机制

Ribonucleases (RNases) play central roles in RNA metabolism and regulation. Recent years, post-transcriptional gene regulation is commonly found in bacterial adaption to environmental stress and that regulated RNA stability and translation mediated by regulated RNA processing and degradation by RNases have shown to be determinant for post-transcriptional control of gene expression. Methanolobus psychrophilus R15 is the model species to study the cold adaption mechanism of archaea. Our previous study found that R15 encodes many cold responsive post-transcriptional regulatory elements, 5′UTR, sRNA, etc. Interestingly, in the mRNA 5′UTR and the intergenic region of polycistronic mRNA, we found many regulatory RNA processing sites, which mediate the cold-responsive regulation of mRNA stabilities and translations in the cold adaption of R15. However, the RNases regulate these key mRNA sites processing and the post-transcriptional regulatory mechanism are still unknown. Our in-depth phylogenomic analyses have shown that β-CASP ribonucleases distributes ubiquitously and highly conserved in Archaea, implying their potentially essential function in RNA processing and degradation in Archaea. In this project, we will thoroughly study the in vitro biochemical activity, in vivo physiological function and the molecular mechanism of archaeal β-CASP ribonucleases, including: 1. Clarify the endonuclease/exonuclease activities and characteristics of archaeal β-CASP RNases, and analyze their structural basis for endonuclease/exonuclease; 2. Explore the key target genes post-transcriptionally regulated by archaeal β-CASP RNases; analyze the important RNA processing sites cleaved by archaeal β-CASP RNases at the single-base resolution and study the post-transcriptional regulatory processes and the molecular mechanism; 3. Analyze the functional proteins interacting with archaeal β-CASP RNases and explore their role and mechanisms in RNA processing and degradation in Archaea. The purpose is to clarify the role and mechanism of archaeal β-CASP RNases function in RNA processing/degradation and post-transcriptional regulation in archaeal environmental adaption, shedding light on these unknown fields in Archaea. Meanwhile, this study will provide theoretical instructions to improve the development and application of cold-adapted methanogen in methane fermentation at low temperature.

核酸酶参与RNA代谢及调控。研究发现核酸酶调控mRNA加工降解，从而调控其稳定性或翻译，在细菌环境适应中功能重要。我们在嗜冷甲烷古菌中发现低温响应的转录后调控元件5′UTR、sRNA等；且在mRNA 5′UTR及多顺反子中发现加工位点，介导其mRNA稳定性及翻译调控，贡献低温响应的基因表达。但参与该过程的核酸酶及分子机制未知。进化分析发现β-CASP核酸酶在古菌中进化保守，暗示其在古菌RNA加工降解中功能重要。本课题拟开展β-CASP核酸酶生理生化功能及作用机制研究，包括：解析其核酸酶活性特征及结构基础；发现其转录后调控的目的基因，单碱基水平解析其RNA加工位点，揭示由此介导的转录后调控过程和机制；发现其互作功能蛋白，探讨古菌RNA加工降解机制。旨在阐明β-CASP核酸酶在古菌RNA加工降解中的功能，揭示其介导的转录后调控过程和机制及在古菌低温适应中的作用，为低温甲烷发酵的应用提供指导。

核酸酶参与RNA代谢及调控，通过调控mRNA的命运—转录、加工降解及翻译，在生命体环境适应中功能重要。本项目基于前期研究中发现古菌中转录后调控及mRNA加工等在低温环境适应中活跃的现象，但参与这些过程的核酸酶及其分子机制均未知，故拟开展古菌中分布广泛且进化保守的β-CASP核酸酶生理生化功能及作用机制研究，目的是阐明古菌关键核酸酶在RNA加工降解及代谢调控中的功能，揭示其介导的转录后调控过程和机制及在古菌低温适应中的作用。目前已取得的主要研究成果如下：1. 首次发现该家族核酸酶aCPSF1为古菌全局转录终止因子，并揭示了其通过3ʹ端切割而介导古菌转录终止的工作模式，该模式也提示了真核转录终止机器由该模式进化而来的衍化路径；2. 解析了该家族核酸酶RNase J的活性特征、结构基础及分子机制，报道了其连续性外切及水解偶联的解链新活性及其分子基础；3. 建立了RNA 5ʹ端测序技术，依托该技术在单碱基水平解析了甲烷古菌组学水平的RNA加工位点图谱，不仅首次发现古菌mRNA也存在加工现象，且揭示了mRNA加工介导古菌多个重要功能蛋白，包括核糖体蛋白等的表达，且同时揭示了核糖体蛋白受转录后调控的新机制； 4. 首次报道了古菌冷保护RNA伴侣蛋白TRAM的生理、生化功能及分子机制，并发现其通过RNA伴侣蛋白活性而抗转录终止从而辅助基因表达并保证最适的转录组组成及菌体的最适生长。这些研究有效推动了对古菌转录后调控关键事件及关键功能核酸酶、伴侣蛋白等的工作机制及分子基础等的认知，取得了原创性且具有国际影响力的研究成果；在该项目的支持下，共发表SCI论文8篇，已按时完成了项目的预期目标。