低温甲烷古菌对冷胁迫的转录后响应机制

Cold-adaptive methanogens play a major role in the methane emission from cold wetlands, yet the cold-adaptive mechanisms remain unknown. One of the severe impairs of lower temperatures on life is causing mRNAs to form higher structures; those would impede the regular turnover of mRNAs, and protein translation. It is known that the bacterial cold-shock proteins (Csp), which function as the RNA chaperones, protect mRNAs from forming secondary structures at cold. The mRNA of CspA from E. coli also exhibits enhanced stability at cold and even folded as a cold-prone translational conformation, which depends on the large 5’untranslated region (5’UTR). So far no confirmed cold-shock protein has been found in Archaea, however, like the CspA mRNA, many mRNAs in methanogenic Archaea possess a large 5’UTR. Previously, we determined that the mRNAs key to methylotrophic methanogenesis in a cold adaptive methanogen showed as long half-life as those of yeast, and the stability was largely determined by its large 5’UTR. This suggests that posttranscriptional regulation may play important roles in the cold adaptation of methanogenic Archaea. . This proposed project will take the psychrophilic methanogen R15 as the study objective. By using the single base-resolution differential RNA deep sequencing (RNA-seq) approach, we will analyze the genome-wide transcription start site (TSS) so that the transcript architecture especially the 5’UTRs, and the global mRNA half-lives in response to cold. Through these analyses we aim to reveal the correlation between mRNA stability and its sequence architecture, so that find the important archaeal “cold box” or “cold motif”. . Following, we will choose the mRNAs for methanogenesis and the putative cold protective proteins (TRAM and DEAD-domain proteins) to test correlations between the mRNA stability, translation efficiency and its sequence architecture through experiments. The in vitro mRNA stability and translation will be analyzed on the mutated transcripts by removing the large 5’UTRs or other “cold-motifs”, so that to confirm the “cold-motifs” and their roles. . Last, we will test the cold protection roles of the putative cold shock proteins (TRAM and DEAD-domain proteins) in a cold sensitive E. coli. Construction of the gene-inactive mutants will be tried for R15. The working mechanisms of the archaeal cold shock proteins will be studied through biochemical analysis as well. . This work will provide an insight into post-transcriptional regulation mechanisms in the cold adaptation of archaea, and the output will provide cold active methanogens and cold protection proteins to biogas fermentation at lower temperatures..

耐冷甲烷古菌驱动低温湿地甲烷的排放，但其冷适应机制未知。低温对生命的威胁是使mRNA形成二级结构，从而影响其正常周转及翻译。细菌冷激蛋白（Csp）保护mRNA低温下的正确构象；CspA的mRNA也因具有长的5’UTR而低温下稳定并被优先翻译。迄今未知古菌的冷激蛋白，但知甲烷古菌的mRNA多具长5’UTR。我们前期证明，产甲烷基因mRNA因长的5’UTR而低温下稳定，说明mRNA结构依赖的转录后调控可能对甲烷古菌冷适应重要。本项目以嗜冷甲烷古菌R15为对象，在组学水平揭示古菌mRNA低温稳定性与序列结构的关系；实验验证产甲烷和抗冷胁迫基因mRNA的低温稳定性及翻译与序列结构的关系，重点是5’UTR-mRNA isoforms的低温稳定性及翻译效率的差异，发现古菌转录后调控重要的mRNA的“冷基序”；发现古菌抗冷蛋白和作用机理。目的是认识转录后调控在古菌冷适应中的作用，并为低温湿地甲烷排放的管理提供思路、为低温沼气发酵提供菌株和功能蛋白。

甲烷古菌是唯一产生大量甲烷的生物。青藏高原低温湿地释放大量甲烷，说明存在低温甲烷古菌，但它们的嗜冷机制未知。该项目以从青藏高原湿地获得的嗜冷甲烷古菌为材料，研究转录后调控在甲烷古菌冷适应中的作用，旨在揭示嗜冷甲烷古菌在低温下活跃的分子基础。研究发现：1) dRNA-seq检测到嗜冷甲烷古菌R15具有组学水平的、响应低温的转录起始位点动态变化，同一个基因产生5’UTR长度不同的转录子；2) dRNA-seq检测到甲烷古菌R15具有全局性的mRNA加工现象，改变了原核生物mRNA无加工的概念。分子生物学实验发现mRNA加工调控古菌核糖体蛋白合成，提出了的转录后调控核糖体组装的新机制；3) dRNA-seq和分子生物学实验发现，甲醇产甲烷途径关键基因的转录本在低温下加工水平提高，并促进蛋白翻译效率，揭示了甲醇产甲烷是低温环境优势途径的分子基础；4)报道了古菌的首个冷休克蛋白TRAM，TRAM具有RNA 伴侣功能，通过转录后调控古菌的转录组及生理学；TRAM帮助细菌耐低温、及植物耐干旱；5)解析了首个古菌核糖核酸酶RNase J与RNA的共晶结构，结合遗传学和生化分析，揭示了其发挥持续性外切的分子机制；6)意外发现古菌中广泛存在的核酸酶aCPSF1行使转录终止功能，结合组学、遗传学及分子生物学手段证明、aCPSF1是首个发现的古菌转录终止因子，它介导的古菌转录终止模式与真核生物Pol II的相似，提供了一个新的遗传学证据，说明真核生物的祖先是古菌；7)嗜冷甲烷古菌R15在实验室传代数年后可在中温生长。结合生理学、生化实验及质谱鉴定，证明S-layer 蛋白N-糖基化是嗜冷甲烷叶菌“进化”为中温菌的基础；8)意外发现甲烷古菌具有二甲基砷脱甲基功能，可用于减少水稻直穗病。项目共发表研究论文10篇。