AbstractThe 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).

摘要中温产甲烷古细菌模式生物Methanosarcina mazei菌株Gö1因其产生甲烷的能力而对气候和环境研究至关重要。在这里，我们在两种生长条件（氮充足和限制）下为M.mazei菌株Gö1建立了Ribo-seq方案。

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.

基于Ribo-seq数据，可以高可信度预测编码蛋白质≤70个氨基酸的93个先前注释的和314个未注释的小ORF的翻译。LC-MS分析验证了62个带注释的小ORF和26个未注释的小ORF的翻译。表位标记，然后进行免疫印迹分析，证实了16个选定的未注释小ORF中13个的翻译。

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 sRNA154, the central regulatory small RNA of nitrogen metabolism.

全面的差异转录和翻译分析表明，314个未注释的小ORF中有29个在转录水平上响应氮可用性而受到差异调节，49个在翻译水平上受到差异调节。大量报道的小RNA正在成为双功能RNA，包括sRNA154，氮代谢的中枢调节小RNA。

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.

几种未注释的小ORF在Methanosarcina物种中是保守的，并且过量产生几种（小ORF编码的）小蛋白质表明了关键的生理功能。总体而言，综合分析为阐明M中大量小蛋白和双功能RNA的功能开辟了一条途径。

mazei..

马泽。。

IntroductionAn unexpected complexity and density of genes within microbial genomes have been revealed by the steadily growing number of sequenced prokaryotic genomes in combination with high throughput OMICS profiling technologies such as next generation sequencing of DNA and RNA and optimized proteomics1,2.

引言微生物基因组中基因的意外复杂性和密度已经通过稳定增长的测序原核基因组数量以及高通量组学分析技术（如DNA和RNA的下一代测序和优化的蛋白质组学）揭示出来1,2。

In addition, discovering genes encoding longer proteins or non-coding RNAs, genome-wide studies have also revealed a potential wealth of small proteins in all kingdoms of life. Small proteins are defined here as ribosomal synthesized proteins of ≤ 70 amino acids (aa) in length that are translated from small open reading frames (sORFs).

此外，发现编码更长蛋白质或非编码RNA的基因，全基因组研究也揭示了生命王国中潜在的大量小蛋白质。小蛋白质在这里定义为长度≤70个氨基酸（aa）的核糖体合成蛋白质，这些蛋白质是从小开放阅读框（SORF）翻译而来的。

They have been frequently overlooked in the past due to various technical and methodological difficulties e.g., in mass spectrometry3,4. Recent emerging tools and evidences demonstrated that small proteins in eukarya, bacteria, and viruses are implicated in important and diverse cellular functions, such as transport, sporulation, signal transduction, virulence, symbiosis or antiCRISPR activity4,5,6,7,8,9,10,11,12.Small proteins in the domain of archaea are underrepresented in recent studies, and only few small proteins are characterized until now13,14.

由于各种技术和方法上的困难，例如在质谱中，它们在过去经常被忽视3,4。最近出现的工具和证据表明，真核生物，细菌和病毒中的小蛋白与重要和多样的细胞功能有关，如运输，孢子形成，信号转导，毒力，共生或抗CRISPR活性4,5,6,7,8,9,10,11,12。古细菌领域的小蛋白在最近的研究中代表性不足，到目前为止只有少数小蛋白被表征13,14。

This occurs to the smaller number of identified and cultivable archaea. Many archaea live in extreme habitats or in symbiosis with multicellular organisms. Consequently, due to the challenging experimental work and prediction tools optimized for bacteria, less is known about small proteins in archaea14.

这发生在数量较少的已鉴定和可培养的古细菌中。许多古细菌生活在极端的栖息地或与多细胞生物共生。因此，由于针对细菌优化的具有挑战性的实验工作和预测工具，对古细菌中的小蛋白质知之甚少14。

Methanosarcina mazei strain Gö1 is an archaeal model organism and represents a methylotrophic methanogenic archaeon15. It produces methane from carbon sources like CO2 plus H2, methanol, methylamine or acetate16,17,18,19 and can fix molecular nitrogen under nitrogen (N) limitation20. The.

Methanosarcina mazei菌株Gö1是一种古细菌模式生物，代表了一种甲基营养型产甲烷古菌15。它从二氧化碳加H2，甲醇，甲胺或乙酸酯等碳源产生甲烷16,17,18,19，并且可以在氮（N）限制下固定分子氮20。的。

To generate a translatome map that provides a comprehensive depiction of translated annotated sORFs and to discover unannotated sORFs in M. mazei Gö1, we optimized the Ribo-seq protocol initially proposed by40,44,45 and further developed it to suit the characteristic of this particular methanoarchaeon under two different growth conditions ( + N and -N) (Fig. 1A for more detailed information).

为了生成一个翻译组图谱，提供翻译注释的sORFs的全面描述，并在M.mazei Gö1中发现未注释的sORFs，我们优化了最初由40,44,45提出的Ribo-seq协议，并进一步开发了它，以适应两种不同生长条件（ + N和-N）下这种特定甲烷动物的特征（图1A提供了更多详细信息）。

The polysome profile analysis confirmed successful capture of translating ribosomes (Fig. 1B, depicted by black lines), followed by conversion of M. mazei polysomes46,47 into monosomes using MNase digestion (Fig. 1B)48,49,50. The effective implementation of Ribo-seq for M. mazei was validated by examining the Ribo-seq coverage of translated ORFs and known non-coding transcripts.

多核糖体谱分析证实成功捕获翻译核糖体（图1B，用黑线描绘），然后使用MNase消化将M.mazei多核糖体46,47转化为单体（图1B）48,49,50。通过检查翻译的ORF和已知的非编码转录本的Ribo-seq覆盖率，验证了Ribo-seq对M.mazei的有效实施。

Exemplarily shown for the protein-coding gene psmB (MM_0694; encoding a proteasome subunit), which exhibited greater cDNA read coverage in the Ribo-seq library as compared to the paired RNA-seq library (Fig. 1C, left panel), indicating translation of this gene. Conversely, the sRNA162, a known non-coding regulatory sRNA51, displayed high read coverage of cDNA in the RNA-seq but no coverage in the Ribo-seq library (Fig. 1C right panel).

示例性地显示了蛋白质编码基因psmB（MM\U 0694；编码蛋白酶体亚基），与成对的RNA-seq文库相比，其在Ribo-seq文库中表现出更大的cDNA读取覆盖率（图1C，左图），表明该基因的翻译。相反，sRNA162（一种已知的非编码调节性sRNA51）在RNA-seq中显示出高的cDNA读取覆盖率，但在Ribo-seq文库中没有覆盖率（图1C右图）。

This observation further validates its non-coding nature. Furthermore, the read coverages of the 5’- and 3’-UTRs of psmB demonstrated higher levels in the RNA-seq library compared to the Ribo-seq library (Fig. 1C, left panel). This indicates that mRNA regions that are not translated or unprotected regions were effectively degraded by MNase, supporting the validity of our Ribo-seq approach.

该观察结果进一步验证了其非编码性质。此外，与Ribo-seq文库相比，psmB的5'-和3'-UTR的读取覆盖率在RNA-seq文库中显示出更高的水平（图1C，左图）。这表明未翻译或未保护区域的mRNA区域被MNase有效降解，支持了我们的Ribo-seq方法的有效性。

Similarly, the operon comprising four small proteins (MM1355-MM1358, TRAM domains) also displayed successful digestion of the 5’- and 3’-UTRs (Fig. 1D). This further confirms the reliabilit.

类似地，包含四种小蛋白（MM1355-MM1358，TRAM结构域）的操纵子也显示出对5'-和3'-UTR的成功消化（图1D）。这进一步证实了可靠性。

Data availability

数据可用性

Ribo-seq and RNA-seq data generated and analyzed during the current study have been deposited in GEO with the accession number GSE240615. The Ribo-seq for M. mazei can be viewed with an interactive online JBrowse instance (http://www.bioinf.uni-freiburg.de/ribobase) on request. All re-processed LC-MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045039, the meta-data is provided in Supplementary Data 10.

在当前研究期间生成和分析的Ribo-seq和RNA-seq数据已保存在GEO中，登录号为GSE240615。可以使用交互式在线JBrowse实例查看M.mazei的Ribo-seq(http://www.bioinf.uni-freiburg.de/ribobase)根据要求。所有重新处理的LC-MS数据都已通过PRIDE合作伙伴存储库存储到ProteomeXchange Consortium，数据集标识符为PXD045039，元数据在补充数据10中提供。

This consists of previously published raw files from the datasets PXD00432556 [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD004325], PXD01979254 [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019792], PXD01199655 [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD011996], as well as datasets PXD055745, and PXD055748, which were produced in house following the methodologies detailed in this manuscript, with publications pending. Source data are provided with this paper..

这包括来自数据集PXD00432556的先前发布的原始文件[https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD004325]，PXD01979254[https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019792]，PXD01199655[https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD011996]，以及数据集PXD055745和PXD055748，这些数据集是按照本手稿中详细介绍的方法在内部生成的，尚待发布。本文提供了源数据。。

Code availability

代码可用性

All the codes regarding functional enrichment analysis are available at the following GitHub repository: https://github.com/RickGelhausen/pathsnake. An archived version of the repository has been generated and is accessible via Zenodo with the following https://doi.org/10.5281/zenodo.13384255.

以下GitHub存储库中提供了有关功能丰富分析的所有代码：https://github.com/RickGelhausen/pathsnake.https://doi.org/10.5281/zenodo.13384255.

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Download referencesAcknowledgementsWe thank Stephanie Färber and Philipp Kible for technical assistance during the establishment of M. mazei Ribo-seq and Rebecca Eulitz during the in vivo validation. We further thank Eva Herdering for providing M. mazei cells grown under different stress conditions for LC-MS/MS analysis.

下载参考文献致谢我们感谢Stephanie Färber和Philipp Kible在体内验证期间建立M.mazei Ribo-seq和Rebecca Eulitz期间提供的技术援助。我们进一步感谢Eva Herdering为LC-MS/MS分析提供了在不同应激条件下生长的M.mazei细胞。

This work was supported by the German Research Foundation (DFG) priority program SPP2002 “Small Proteins in Prokaryotes, an Unexplored World” (Grants SH580/7-1 and SH580/7-2 to C.M.S., CIBSS-EXC-2189-390939984 and BA2168/21-2 to R.B., TH 872/10-1 and 10-2 to A.T., RSCHM1052/20-1 + 20-2, RSCHM1052/19-2 to R.A.S.).

这项工作得到了德国研究基金会（DFG）优先项目SPP2002“原核生物中的小蛋白质，未探索的世界”的支持（授予C.M.S.的SH580/7-1和SH580/7-2，授予CIBSS-EXC-2189-390939984和BA2168/21-2给R.B.，授予A.T.的TH 872/10-1和10-2，授予R.A.S.的RSCHM1052/20-1 + 20-2，RSCHM1052/19-2）。

This work was supported by the BMBF-funded de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI) (031A532B, 031A533A, 031A533B, 031A534A, 031A535A, 031A537A, 031A537B, 031A537C, 031A537D, 031A538A).FundingOpen Access funding enabled and organized by Projekt DEAL.Author informationAuthors and AffiliationsInstitute for General Microbiology, Kiel University, 24118, Kiel, GermanyMuhammad Aammar Tufail, Britta Jordan, Tim Habenicht, Miriam Gutt, Lisa Hellwig & Ruth A.

这项工作得到了德国生物信息学基础设施网络（de.NBI）内由BMBF资助的de.NBI云（031A532B、031A533A、031A533B、031A534A、031A535A、031A537A、031A537B、031A537C、031A537D、031A538A）的支持。资金开放获取资金由Projekt交易启用和组织。作者信息作者和附属机构基尔大学普通微生物学研究所，24118，基尔，GermanyMuhammadAammarTufail，BrittaJordan，TimHabenicht，MiriamGutt，LisaHellwig＆Ruth A。

SchmitzInstitute of Molecular Infection Biology, University of Würzburg, 97080, Würzburg, GermanyLydia Hadjeras & Cynthia M. SharmaBioinformatics Group, Department of Computer Science, University of Freiburg, 79110, Freiburg, GermanyRick Gelhausen & Rolf BackofenSystematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Kiel University, 24105, Kiel, GermanyLiam Cassidy & Andreas TholeyAuthorsMuhammad Aammar TufailView author publicationsYou can also search for this author in.

维尔茨堡大学SchmitzInstitute of Molecular Infection Biology，维尔茨堡大学，97080，维尔茨堡，GermanyLydia Hadjeras＆Cynthia M.SharmaBioinformatics Group，弗莱堡大学计算机科学系，79110，弗莱堡，GermanyRick Gelhausen＆Rolf BackofenSystematic Proteome Research＆Bioanalytics，基尔大学实验医学研究所，24105，基尔，GermanyLiam Cassidy＆Andreas TholeyAuthorsMuhammad Aammar TufailView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsM.A.T., B.J., and L.H. contributed equally and are co-sharing first authors. C.M.S., R.B., and R.A.S. initiated the project. L.H., C.M.S., and R.A.S. designed the experiments. B.J. and M.G. cultured M. mazei and harvested cell pellets for Ribo-seq under L.H.

PubMed谷歌学术贡献。A、 。C、 M.S.，R.B。和R.A.S.启动了该项目。五十、 H.，C.M.S.和R.A.S.设计了实验。B、 J.和M.G.在L.H.下培养M.mazei并收获Ribo-seq的细胞沉淀。

supervision. L.H. established Ribo-seq for M. mazei. B.J., M.A.T., and L.H. manually curated the predicted unannotated sORFs, the annotated sRNAs, suspected mis-annotated genes, and consolidated the data from different genome annotations including the newest annotation. B.J. performed the conservation analyses, predicted the function of unannotated small proteins, and performed the cloning and western blot analyses together with L.He.

监督。五十、 H.为M.mazei建立了Ribo-seq。B、 J.，M.A.T。和L.H.手动策划了预测的未注释的SORF，注释的sRNA，疑似注释错误的基因，并整合了来自不同基因组注释（包括最新注释）的数据。B、 J.进行了保守性分析，预测了未注释的小蛋白的功能，并与L.He一起进行了克隆和蛋白质印迹分析。

R.G. performed the bioinformatic processing of the Ribo-seq data, established a workflow for differential expression analysis for Ribo-seq data, created an annotation database for M. mazei and developed a script for functional enrichment analysis. M.A.T. further analysed the differential expression data and did functional enrichment analysis and visualization.

R、 G.对Ribo-seq数据进行了生物信息学处理，建立了Ribo-seq数据差异表达分析的工作流程，为M.mazei创建了注释数据库，并开发了功能富集分析的脚本。M、 A.T.进一步分析了差异表达数据，并进行了功能富集分析和可视化。

B.J. and M.A.T. did the data visualization under the supervision of R.A.S. T.H. conducted the structure prediction analysis with AlphaFold 2. L.H. and R.G. created a JBrowse instance for the described Ribo-seq datasets in the RIBOBASE database to facilitate their accessibility to the scientific community.

B、 J.和M.A.T.在R.A.S.T.H.的监督下进行了数据可视化。使用AlphaFold 2进行了结构预测分析。五十、 H.和R.G.为RIBOBASE数据库中描述的Ribo-seq数据集创建了一个JBrowse实例，以便于科学界访问。

L.C. and A.T. performed all LC-MS/MS analyses including interpretation. B.J., M.A.T., and L.H. wrote a first draft of the manuscript. B.J., M.A.T., L.He. and R.A.S. finalized writing the manuscript. B.J., M.A.T., and R.A.S. incorporated the changes according to the feedback from all the authors. R.B., C.M.S., and R.A.S.

五十、 C.和A.T.进行了所有LC-MS/MS分析，包括解释。B、 J.，M.A.T。和L.H.撰写了手稿的初稿。B、 J.，M.A.T.，L.He。和R.A.S.完成了手稿的撰写。B、 J.，M.A.T。和R.A.S.根据所有作者的反馈纳入了这些变化。R、 B.，C.M.S。和R.A.S。

supervised the research and provided resources and f.

监督研究并提供资源和f。

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Nature Communications thanks Jorg Soppa, Rani Baes and the other, anonymous, reviewer for their contribution to the peer review of this work. A peer review file is available.

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Reprints and permissionsAbout this articleCite this articleTufail, M.A., Jordan, B., Hadjeras, L. et al. Uncovering the small proteome of Methanosarcina mazei using Ribo-seq and peptidomics under different nitrogen conditions.

转载和许可本文引用本文Tufail，M.A.，Jordan，B.，Hadjeras，L。等人在不同的氮条件下使用Ribo-seq和肽组学揭示了甲烷八叠球菌的小蛋白质组。

Nat Commun 15, 8659 (2024). https://doi.org/10.1038/s41467-024-53008-8Download citationReceived: 08 October 2023Accepted: 25 September 2024Published: 06 October 2024DOI: https://doi.org/10.1038/s41467-024-53008-8Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

。https://doi.org/10.1038/s41467-024-53008-8Download引文接收日期：2023年10月8日接收日期：2024年9月25日发布日期：2024年10月6日OI：https://doi.org/10.1038/s41467-024-53008-8Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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