Institute for General Microbiology, Kiel University, 24118, Kiel, Germany.
Institute of Molecular Infection Biology, University of Würzburg, 97080, Würzburg, Germany.
Nat Commun. 2024 Oct 6;15(1):8659. doi: 10.1038/s41467-024-53008-8.
The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1 is crucial for climate and environmental research due to its ability to produce methane. Here, we establish a Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency and limitation). The translation of 93 previously annotated and 314 unannotated small ORFs, coding for proteins ≤ 70 amino acids, is predicted with high confidence based on Ribo-seq data. LC-MS analysis validates the translation for 62 annotated small ORFs and 26 unannotated small ORFs. Epitope tagging followed by immunoblotting analysis confirms the translation of 13 out of 16 selected unannotated small ORFs. A comprehensive differential transcription and translation analysis reveals that 29 of 314 unannotated small ORFs are differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. A high number of reported small RNAs are emerging as dual-function RNAs, including sRNA, the central regulatory small RNA of nitrogen metabolism. Several unannotated small ORFs are conserved in Methanosarcina species and overproducing several (small ORF encoded) small proteins suggests key physiological functions. Overall, the comprehensive analysis opens an avenue to elucidate the function(s) of multitudinous small proteins and dual-function RNAs in M. mazei.
嗜中温产甲烷古菌模式生物 Methanosarcina mazei 菌株 Gö1 因其产甲烷能力而成为气候和环境研究的关键。在这里，我们建立了一种用于 M. mazei 菌株 Gö1 在两种生长条件（氮充足和限制）下的 Ribo-seq 方案。基于 Ribo-seq 数据，可高度置信地预测 93 个先前注释和 314 个未注释的小 ORF（编码≤70 个氨基酸的蛋白质）的翻译。LC-MS 分析验证了 62 个注释小 ORF 和 26 个未注释小 ORF 的翻译。通过表位标记和免疫印迹分析确认了 16 个选定的未注释小 ORF 中有 13 个被翻译。综合差异转录和翻译分析表明，在转录水平上，314 个未注释的小 ORF 中有 29 个对氮可用性有差异调节，在翻译水平上有 49 个对氮可用性有差异调节。大量报道的小 RNA 正在作为双功能 RNA 出现，包括 sRNA，氮代谢的中央调控小 RNA。几个未注释的小 ORF 在 Methanosarcina 物种中保守，并且过量产生几个（小 ORF 编码的）小蛋白表明具有关键的生理功能。总体而言，综合分析为阐明 M. mazei 中众多小蛋白和双功能 RNA 的功能开辟了一条途径。
