Isotope-based analysis of modified tRNA nucleosides correlates modification density with translational efficiency.
Isotope-based analysis of modified tRNA nucleosides correlates modification density with translational efficiency.
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DOI：
10.1002/anie.201203769
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发表时间：
2012-10-29
影响因子：
16.6
通讯作者：
Carell, Thomas
中科院分区：
文献类型：
--
作者：
Brandmayr, Caterina;Wagner, Mirko;Brueckl, Tobias;Globisch, Daniel;Pearson, David;Kneuttinger, Andrea Christa;Reiter, Veronika;Hienzsch, Antje;Koch, Susanne;Thoma, Ines;Thumbs, Peter;Michalakis, Stylianos;Mueller, Markus;Biel, Martin;Carell, Thomas
转移RNA（tRNA）是将遗传信息翻译成肽序列所需的衔接分子。[1]在核糖体上，每个tRNA的反密码子读取信使RNA的相应密码子。这种反密码子-密码子的相互作用使得核糖体大亚基催化氨酰-tRNA 3 ′末端的同源氨基酸与生长中的肽链之间的酰胺键形成。[2]这一过程所需的tRNA接头显示出惊人的化学多样性。[3]除了四种典型的核苷A、C、G和U外，100多种修饰的核苷是关键成分（图1）。[4]在反密码子茎环中发现了最多样和最复杂的化学结构，无论是在反密码子的摆动位置还是直接邻近反密码子的3位置，[5]这表明化学复杂性是建立翻译保真度所必需的。[6]核糖体似乎需要经过修饰的反密码子区域来更好地区分正确配对的tRNA与错误配对的相互作用，以阻止例如可能导致移码的密码子滑动过程。[7]为了研究核苷修饰的集合如何影响翻译效率，我们通过基于同位素稀释的LC-MS方法分别定量各种组织中的tRNA修饰。（有关材料和方法的详细信息见支持性信息、表S1和图S1）。通过体外翻译系统将定量修饰水平与翻译效率相关联。在实验中，11种代表性的tRNA修饰（图1）被化学合成为同位素标记的衍生物。[8]大多数研究的核苷位于延伸的反密码子内，[9]其他合成的修饰核苷位于其他位置。[10]作为用于分析的生物材料，我们从小鼠和猪中选择了一系列不同的器官组织。使用猪组织是因为其可大量获得，而在后期分析鼠组织以确认在遗传上更明确的微生物中的结果。对于猪，从每种器官中采集5-10 g来自两只动物的组织，而鼠样品从两组5只动物中获得，分析其整个器官。在总tRNA提取和完全酶水解成核苷后，加入同位素标记的tRNA修饰的混合物，并对溶液进行LC-MS分析。测定了天然核苷与同位素标记核苷的质量峰积分比值，并测定了先前测定的各研究修饰核苷的校准曲线，然后允许对相应修饰进行精确平行定量（见支持性信息中的图S2）。[8a]至少一式三份进行LC-MS定量，并对各组织的结果取平均值。通过这种方式，实验的误差范围被限制在5%左右。小鼠和猪样品的定量值分别在图2A和图B中以颜色编码显示，以及tRNA序列中测量的修饰的近似位置。这些值代表每1000个tRNA分子中每种修饰的测量数量（%）（精确值见支持信息中的表S2 -7）。因此，数据不是得出给定组织中修饰的绝对浓度，而是直接显示分析的tRNA组被修饰的程度。对于代表性的鼠和猪组织，额外的定量...
Transfer RNAs (tRNAs) are adapter molecules needed to translate genetic information into a peptide sequence.[1] At the ribosome, the anticodon of each tRNA reads the corresponding codon of the messenger RNA. This anticodon–codon interaction allows the ribosome s large subunit to catalyze amide-bond formation between the cognate amino acids present at the 3о terminus of aminoacyl-tRNAs and the growing peptide chain.[2] The tRNA adapters required for this process display a surprisingly large chemical diversity.[3] Aside from the four canonical nucleosides A, C, G, and U, more than 100 modified nucleosides are key constituents (Figure 1).[4] The most diverse and complex chemical structures are found in the anticodon stem-loop either in the anticodon at the wobble position or directly adjacent to the 3о position of the anticodon,[5] suggesting that here the chemical complexity is necessary to establish translational fidelity.[6] The ribosome seems to need the modified anticodon region to better distinguish correctly base-paired tRNA from mispaired interactions in order to prohibit, for example, codon-slippage processes that would lead to frameshifts.[7] In order to investigate how the set of nucleoside modifications influences the translational efficiency we quantified the tRNA modifications individually in various tissues by an isotope-dilution-based LC–MS method.(Details on the materials and methods are given in the Supporting Information, Table S1, and Figure S1). The quantified modification levels were correlated with the translational efficiency by means of an in vitro translation system. For the experiments, 11 representative tRNA modifications (Figure 1) were chemically synthesized as isotope-labeled derivatives.[8] A majority of the investigated nucleosides are located inside the extended anticodon,[9] the other synthesized modified nucleosides are found at various other positions.[10] As biological material for the analysis we chose a range of different organ tissues from mouse and pig. Porcine tissues were used because they are available in large amounts, while murine tissues were analyzed at a later stage to confirm the results in a genetically more defined organism. For pig, 5–10 g of tissue from two animals was sampled from each organ, while murine samples were obtained from two sets of five animals of which whole organs were analyzed. After total tRNA extraction and complete enzymatic hydrolysis to nucleosides, a mixture of the isotope-labeled tRNA modifications was added and the solution was subjected to LC–MS analysis. The ratios of the mass peak integrals from natural to isotope-labeled nucleosides were determined and calibration curves, which were previously measured for each investigated modified nucleoside, then allowed exact parallel quantification of the respective modifications (see Figure S2 in the Supporting Information).[8a] LC–MS quantification was performed at least in triplicate and results were averaged for each tissue. The error margin of the experiment was in this way limited to around 5%.The obtained quantitative values for mouse and pig samples are shown color coded in Figure 2A and B, respectively, together with the approximate positions of the measured modifications in the tRNA sequence. The values represent the measured number of each modification per 1000 tRNA molecules (%)(exact values are listed in TablesS2–7 in the Supporting Information). Therefore, rather than yielding the absolute concentration of a modification in a given tissue, the data show directly the extent to which the analyzed tRNA set is modified. For representative murine and porcine tissues, an additional quantification of …
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影响因子：
4.5
作者：
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