王恩多院士两篇JBC文章解析tRNA新分子机制

　　

生物通报道：tRNA是细胞内主要的RNA之一，是蛋白质合成中的关键生物大分子。在所有胞内的RNA中，tRNA具有最多的修饰，这些修饰对于tRNA在细胞内发挥功能起着重要作用，缺失某些修饰将引起细胞的严重缺陷甚至导致人类疾病。

近期来自中科院上海生科院的王恩多研究组通过质谱、定点突变和酶学动力学等生物化学手段鉴定出tRNA上参与人NSun6（hNSun6）识别的关键元件。这一研究成果公布在Journal of Biological Chemistry杂志上。
通常tRNA氨基酸接受茎部分的核苷酸较少被修饰，第72位的5-甲基胞嘧啶（m5C）修饰是为数不多的几个修饰之一。该位点的m5C修饰仅存在于高等真核生物中且极为保守，提示这个修饰可能在高等真核生物中具有重要的功能。NSun家族成员是真核生物中最主要的RNA m5C甲基转移酶。目前，Nsun6已经被报道能够催化tRNA第72位的甲基化修饰，但其对tRNA识别的关键元件尚不清楚。

在这篇文章中，研究人员通过质谱、定点突变和酶学动力学等生物化学手段鉴定出tRNA上参与人NSun6（hNSun6）识别的关键元件。研究发现hNSun6的识别依赖于tRNA的倒L型三级结构，以及位于tRNA接受茎和D茎的特定的碱基或碱基对。具体识别元件为：（1）全长且正确折叠的tRNA；（2）3’端的CCA末端以及催化位点C72；（3）接受茎上的U73；（4）接受茎第二和第三对碱基对；（5）D茎的11:24和12:23两对碱基对。研究结果表明hNSun6采用三级结构和某些tRNA分子上的特定的元件这一复杂网络识别RNA。其它细胞内的 RNA似乎都不能满足hNSun6 识别的所有需要的条件，因此可以认为hNSun6是专一于tRNA的甲基转移酶。

此外，近期这一研究组也同样在JBC杂志上发文，鉴定了细菌来源的多种ThrRS在N1结构域的存在与N2结构域活性位点的分布上的多样性与复杂性。

蛋白质合成中，氨基酰-tRNA合成酶（AARS）从源头上介导氨基酸与tRNA之间的精确匹配，生成正确的氨基酰-tRNA，为核糖体提供原料。一方面，AARS需要高效地催化氨基酰化反应以合成正确的氨基酰-tRNA，满足蛋白质合成；另一方面，AARS需要通过水解编校功能以确保氨基酸与tRNA的正确匹配。

苏氨酰-tRNA合成酶（ThrRS）属于经典的具有编校功能的AARS，包含N1结构域（功能未知）、N2编校结构域、氨基酰化结构域与RNA结合结构域。N1结构域在氨基酰化、编校反应中的功能一直未知。

王恩多研究组通过基因组数据分析鉴定了细菌来源的多种ThrRS在N1结构域的存在与N2结构域活性位点的分布上的多样性与复杂性。通过体内与体外实验证明了多种细菌类ThrRS介导的蛋白质合成质量控制机制，揭示了发生在细菌类ThrRS N2结构域的编校功能依赖于N2与N1结构域间的通讯机制。进一步的，鉴定了N1与N2结构域中调控结构域间通讯的氨基酸残基与发挥作用的途径。该研究阐明了ThrRS N1结构域在遗传信息精确性传递中的关键作用及其发挥作用的分子机制；如果破坏N1-N2结构域的通讯机制产生错误翻译（mistranslation），则导致细菌与微生物死亡。

原文摘要：
Translational Quality Control by Bacterial Threonyl-tRNASynthetases

Translational fidelity mediated by aminoacyl-tRNA synthetases ensures the generation of the correct aminoacyl-tRNAs, which is critical for most species. Threonyl-tRNA synthetase (ThrRS) contains multiple domains, including an N2 editing domain. Of the ThrRS domains, N1 is the last to be assigned a function. Here, we found that ThrRSs from Mycoplasma species exhibit differences in their domain composition and editing active sites compared with the canonical ThrRSs. The Mycoplasma mobile ThrRS, the first example of a ThrRS naturally lacking the N1 domain, displays efficient post-transfer editing activity. In contrast, the Mycoplasma capricolum ThrRS, which harbors an N1 domain and a degenerate N2 domain, is editing-defective. Only editing-capable ThrRSs were able to support the growth of a yeast thrS deletion strain (ScΔthrS), thus suggesting that ScΔthrS is an excellent tool for studying the in vivo editing of introduced bacterial ThrRSs. On the basis of the presence or absence of an N1 domain, we further revealed the crucial importance of the only absolutely conserved residue within the N1 domain in regulating editing by mediating an N1-N2 domain interaction in Escherichia coli ThrRS. Our results reveal the translational quality control of various ThrRSs and the role of the N1 domain in translational fidelity.
Sequence-specific and Shape-selective RNA Recognition by the Human RNA m5C Methyltransferase NSun6

Human NSun6 is an RNA methyltransferase that catalyses the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to C72 of tRNAThr and tRNACys. In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well-folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.