Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis
题目：通过 tRNA 高通量测序和修饰分析进行微生物组学表征
作者及单位：
Michael H. Schwartz， Haipeng Wang， […], Tao Pan&A. Murat Eren
Tao Pan
- Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA
- Committee on Microbiology, University of Chicago, Chicago, IL, 60637, USA
A. Murat Eren
- Committee on Microbiology, University of Chicago, Chicago, IL, 60637, USA
- Department of Medicine, University of Chicago, Chicago, IL, 60637, USA
- Marine Biological Laboratory, Woods Hole, MA, 02543, USA
发表期刊及时间：
Nature Communicationsvolume 9, Article number: 5353 (2018) Published: 17 December 2018
摘要：
Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with ==protein translation dynamics==（此处我认为可以理解为蛋白质翻译的动态过程）. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are ==phylogenetically tractable==（此处我认为可以理解为进化上保守）, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific ==in situ==（原位的） insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.
高通量测序的发展促进了对天然微生物的多样性和功能的深刻洞察; 然 而，目前的测序策略不足以揭示与蛋白质翻译动态过程相关的微生物群落的生理状态。 转运 RNA（tRNA）是蛋白质合成机器的核心组成部分，存 在于所有活细胞中，并且在进化上是保守的，因此易于追溯，这使它们成为获得对环境微生物的生理学洞察的理想目标。 在这里，我们报告了直接测序方法--tRNA-seq 和软件套件--tRNA-seq-tools，以复原微生物 tRNA 转录物的序列，丰度谱和转录后修饰。 我们使用tRNA‒seq分析盲肠样品区分高脂肪和低脂肪喂养的小鼠，并将该方法与利用16S核糖体RNA基因扩增子的方式相比较，揭示了tRNA修饰中的分类和饮食依赖性变异。我们的结果提供 了对复杂环境微生物组内 tRNA 基因表达和转录后修饰的动态的分类群 特异性原位洞察。
Fig. 1 tRNA modifications of bacterial cultures. Red and black lines show mutation fractions in representative tRNA sequences with (+DM) and without (-DM) demethylase treatment, respectively. a E. coli tRNAPro(CGG) shows m1G37 and s4U8. b E. coli tRNAPhe(GAA) shows acp3U47, ms2i6A37 and s4U8. The additional peak denoted by asterisk (*) may represent an unknown modification. c B. subtilis tRNASer(UGA) shows m1A22, and s4U8. d B. subtilistRNAGlu(UUC) shows m1A22. e S. aureus tRNALeu(UAG) shows m1G37, m1A22 and s4U8. f S. aureus tRNASer(GCU) shows m1A22 and s4U8. g B. viscericola tRNAArg(ACG) shows m1G37 and I34
图一 细菌培养物的 tRNA 修饰。红色和黑色线分别表示具有（+ DM）和不含（-DM）去甲基化酶处理的代表性 tRNA 序列中的 突变部分。 a. 大肠杆菌脯氨酸 tRNA（CGG） 展示出 m1G37 and s4U8 修饰。 b. 大肠杆菌 苯丙氨酸 tRNA（GAA）显示 acp3U47, ms2i6A37 and s4U8 修饰。 由星号（*）表示的附加峰值可表示未 知修饰。 c. 枯草芽孢杆菌丝氨酸 tRNA（UGA）显示 m1A22 和 s4U8 修饰。 d.枯草芽孢杆菌谷氨酸 tRNA（UUC）显示 m1A22 修 饰。 e.金黄色葡萄球菌的亮氨酸 tRNA （UAG） 显示出 m1G37, m1A22 and s4U8 修饰。f.金黄色葡萄球菌的丝氨酸 tRNA（GCU） 显示出 m1A22 and s4U8 修饰。 g. B.viscericola（Barnesiella viscericola） 精氨酸 tRNA（ACG）显示 m1G37 和 I34 修饰
Fig 2. Mutation fractions of two tRNA sites. Heatmaps of mutation fractions for positions 22 and 8 (using standard, canonical tRNA nomenclature) are shown. tRNAs with different anticodons are grouped by their sequences at the respective position of modification (in parenthesis) and in alphabetical orders. Only E. coli and B. subtilis tRNA modifications have been mapped previously by 2D-TLC and LC/MS, but the mapping was not done for every tRNA species12. Every E. coli and B. subtilis tRNA species with mutation fraction at10-times above background is marked with a circle on the right with the following designations: Purples correspond to those known to be present and also identified by sequencing here; blacks correspond to those supposed to be absent but identified by sequencing; greens correspond to those not mapped previously but identified by sequencing; oranges correspond to those known to be present but were not found by sequencing. a m1A22; R corresponds to A or G. b s4U8
图 2。 两个 tRNA 位点的突变分数。位置 22 和 8（使用标准、权威 tRNA 命名法） 突变分数 的热力图在图中被画出。不同反密码子的 tRNA 按照他们各自修饰的位置（在圆括号中）和 字母顺序被分组。过去只有 E.coli 和 B. subtilis的 tRNA 修饰被 2D-TLC 和 LC/MS 对比，但 并没有对所有的 tRNA 种类进行比对。每一个 E.coli 和 B. subtilis中 tRNA 种类， 突变分数超 过背景 10 倍时会在右边用圆圈标记出来。圆圈这样设计：已知会出现并且也被测序识别的 用紫色标记；已知会出现但并没有被测序识别出的用黑色标记；过去没有比对但被测序识别 出的用绿色标记；已知会出现但测序时没有发现的用橙色标记。a m1A22；R 对应于 A 或 G。 b s4U8。
Fig 3. Microbiome tRNA-seq workflow and taxonomy analysis. a Workflow of tRNA sequencing of gut microbiome samples fed with a high-fat (HF) or low-fat (LF) diet and ==de novo==（从头分析） tRNA assignment. Conserved tRNA residues that were searched for in this work are shown in red. b Dendrograms compare relationships between HF and LF samples that were inferred based on community profiles of tRNA transcripts, or 16S rRNA gene amplicons. c Class-level taxonomy for averaged HF and LF samples based on tRNA-seq (top) and 16S rRNA gene amplicons (bottom). All bacterial classes at >1% level are shown in distinct colors, all other bacterial classes are grouped together and shown in purple. d tRNAGly taxonomy for anticodons GCC, UCC, and CCC. e tRNAGlu taxonomy for anticodons UUC and CUC. Among the other category for GCC/UCC/CCC and UUC, no class has an abundance of ≥1%; for CUC, other classes with an abundance of ≥1% include Alphaproteobacteria, Gemmatimonadetes, and Ignavibacteria. tRNAs decoding these two amino acids are the most abundant in our tRNA-seq results
图3. 微生物组tRNA-seq工作流程以及分类学分析 a 用高脂肪饮食组（HF）或低脂肪饮食组（LF）的肠道微生物组样品的tRNA测序以及tRNA全新匹配的工作流程。此工作中寻找到的保守tRNA残基被标注为红色。 b 系统树图，比较HF组和LF租两个样品的关系，这些关系基于tRNA转录物或16S rRNA基因扩增子的群组特征推断得到。 c 基于tRNA‒seq（上图）和16S rRNA基 因扩增子（下图）的平均HF和LF样品的类级分类。所有> 1％水平的细菌类别以不同的颜色显示，所有其他细 菌类别组合在一起并以紫色显示。 d 甘氨酸tRNA反密码子GCC, UCC, 和 CCC 的分类. e 谷氨酸tRNA反密码 子UUC和CUC的分类。在GCC / UCC / CCC和UUC的其他类别中，没有一个类别的丰度≥1％；对于CUC，其他丰度≥1％的类别包括甲型变形菌（Alphaproteobacteria），芽单胞菌（Gemmatimonadetes）和 Ignavibacteria（无中文名）。在我们的tRNA‒seq结果中这两种氨基酸的tRNA是最丰富的。
==de novo==（从头分析）：不依赖于已有的注释文件全新分析
Fig 4. Microbiome tRNA modification analysis. a Workflow for modification assignment using mutation ==signatures==（信号）. b–e Representative positional plots showing m1A and s4U modifications for transcripts of tRNASer(GCU) (b), tRNASer(UGA) (c), tRNASer(GGA) (d), and tRNASer(CGA) (e), HF-fed mouse sample. The peak numbers correspond to those described in the text with peak 1 called for s4U8 and peak 2 for m1A22. Peak 3 is located around nucleotide 73–79 in the type II tRNASers, but is m1A59 in the standard tRNA nomenclature. Red and black lines show mutation fractions in tRNASer sequences with (+DM) and without (−DM) demethylase treatment, respectively
图 4 微生物组 tRNA 修饰分析。 A. 使用变异信号进行修饰分配的工作流程。 B-E. 代表性的位点图展示了 HF 喂食小鼠样本 tRNA Ser（GCU）(b), UGA(c),GGA(d),CGA(e)） 转录本的 m1A 和 s4U 修饰。峰的数量与文中描述的相当，峰 1 为 s 4 U8，峰 2 为 m 1 A22 。峰 3 定位在二 型 tRNA Sers 核苷酸 73-79 周围，但在标准 tRNA 系统命名中却是 m 1 A59 。红色和黑色的线 分别展示了 tRNA 测序中有（+DM） 和没有（-DM） 去甲基化酶处理的变异部分
Fig 5. Taxonomic differences of modification sites. a Examples of aligning tRNA sequencing reads to two seed sequences of tRNASer(UGA) from Lactobacillus, class Bacilli, and Bifidobacterium, class Actinobacteria without and with demethylase treatment. Modification sites identified (s4U and m1A) are highlighted between the white lines. bm1A22, m1A58/59, and s4U8 identified in the abundant bacterial classes from Fig. 3c in the context of their phylogenetic relationship. Large fonts indicate bacterial classes in which the majority of the modifications are found (m1A22 in Clostridia and Bacilli, m1A58/59 in Actinobacteria, and s4U8 in Clostridia and Bacilli). c Proximal location of the m1A22 (red), m158 (blue), and m159 (green) modifications in a tRNA three-dimensional structure
图5. a. 去甲基化酶处理会使得 修饰丢失，其中 和 修饰是在多物种间都存在的 b. 图3c中各类细菌的进化关系及其所包含的修饰类型 c. 三维结构展示不同修饰位点
==个人比较认可这种方式，用自己的话把图表描述一下，不用翻译其中的每一句话，而是解释图表的横纵坐标或线条的意思，更容易理解==
Fig 6. Comparisons of mutation fractions of HF versus LF samples. Bacterial families with the highest numbers of modifications at the respective nucleotides are shown. Each pair shows HF and LF samples with distinct anticodons marked on top. The amino acid whose codons are read by the corresponding tRNA with designated anticodon can be found in Supplementary Table 4. Box-and-whisker plots show median as a line, upper and lower quartiles in the box, and outliers outside of the line. a m1A22 from Lachnospiraceae, class Clostridia. b m1A58 and m1A59 from Bifidobacteriaceae, class Actinobacteria. c s4U8 from Lachnospiraceae, class Clostridia
图6. HF 和 LF 样品突变分数的比较。 显示了在各自的核苷酸上修饰最多的细菌科。 每一对都显 示了顶部标记有不同反密码子的 HF 和 LF 样品。 其密码子由指定反密码子的相应 tRNA 读 取的氨基酸见补充表 4。 箱线图以直线表示中间值，盒中的上、下四分位数，以及线外的异 常值。 a： 来自 Lachnospiraceae, class Clostridia 的 m1A22。 b： m1A58 和 m1A59 来自 Bifidobacteriaceae, class Actinobacteria.。 c： 来自 Lachnospiraceae, class Clostridia 的 s4U8
Fig. 7. Analysis of differential protein expression and tRNA modification. Proteomics data from HF- and LF-fed mice were from reference36. a Average differential expression of 849 proteins between HF- and LF-fed mouse gut microbiome from day 43 mice that most mimics the experimental condition of our tRNA-seq experiment. b BLASTp protein sequence analysis shows that most of these proteins are from class ==Clostridia（梭菌 ）==. c Quantitative difference between the clostridia proteins from day 43 mice. Lines show the boundaries of the proteins used for downstream analysis that are highly enriched (log >1, 88 proteins) or depleted (log< −1, 105 proteins) in HF over LF samples. The difference in amino acid (d) or codon content (e) determined by subtracting the compositions of HF over-expressed proteins minus the HF under-expressed proteins. The amino acids or codons for which their decoding tRNAs were found to contain m1A modifications are in red: Cys, Glu, Gln, Ser. The difference in amino acid pair (f) and codon pair content (g) determined by subtracting the pair compositions of HF over-expressed proteins minus the HF under-expressed proteins. The first amino acid represents the N-terminal residue and the first codon represents the 5’ codon
图7. 差异的蛋白表达和 tRNA 修饰的分析。a 高脂和低脂喂养小鼠 43 天后， 其肠微生物组中 849 个蛋白的平均差异表达，第 43天的实验条件和本文 tRNA-seq实验的条件最相似。 b 蛋白序列比对分析显示测得的大多数蛋白来源于 Clostridia 梭菌类。 c 两列黑线表示用于后续分析的蛋白挑选分界线： log >1 有 88 个 蛋白， 在 HF 中高丰度。 log < -1,105 个蛋白， HF 中低丰度。 log 是 基于高脂与低脂喂养小鼠样本的比值， 通过减去 HF 过表达蛋白的组成、减去 HF 低表达蛋白的组成从而 确定氨基酸(d)或密码子含量(e)的差异。 一些氨基酸或密码子相应的 tRNAs 被发现含有 m1A 修饰， 这些氨基酸或密码子标为红色： Cys、 Glu、 Gln、 Ser。 通过减去 HF 过表达蛋白和 HF 低表达蛋白的配对组成， 确定了氨 基酸对(f)和密码子对(g)含量的差异。 f 中横坐标 first amino acid 代表 N-末端残基， g 中横坐标 first codon 代表 5’ 密码子。
==个人比较认可这种方式，用自己的话把图表描述一下，不用翻译其中的每一句话，而是解释图表的横纵坐标或线条的意思，更容易理解==
翻译小组：
李碧琪、陈凯星、叶名琛、郑易民、王俊豪、倪豪辰、黄敬潼、黄子亮、邓峻玮、常彦琪、郑凌伶
