Fluolab【JACS】揭秘tRNA修饰：首个可实现细胞内Queuine空间分布成像的新技术，成像对比度提升达3倍
文章标题： Enzyme-Mediated Covalent Labeling Enables In Situ Imaging of RNA Modification States
通讯作者： Neal K. Devaraj
文章概要
引言
在复杂的细胞生命活动中，转录后tRNA修饰扮演着基因表达调节者的关键角色，但由于缺乏有效的细胞内原位追踪手段，科学家对其空间分布和动态调节的理解一直受限。传统的测序和生化分析虽然能提供群体水平的数据，但必须提取RNA，这一过程无情地抹去了珍贵的空间信息并掩盖了细胞间的异质性。特别是Queuine（Q碱基） 这种独特的超修饰碱基，它广泛存在于真核生物tRNA的摆动位置，影响着翻译的准确性、应激反应及多种生理病理过程。针对这一挑战，加州大学圣地亚哥分校的Neal K. Devaraj团队开发了一种创新的化学酶法RNA荧光标记策略，首次实现了在哺乳动物细胞中对tRNA特定修饰状态的高分辨率空间分析。
Figure 1. Chemoenzymatic labeling enables relative analysis of queuine modification levels on tRNAs. (A) Graphical representation of the method used to label queuine-unmodified tRNAs in samples. (B) Total RNA samples run on a denaturing gel after labeling with preQ1-Cy5 and E. coli TGT. The gel was stained with a nonspecific nucleic acid dye to ensure equal loading and to determine the molecular weight of the labeled products, as shown on the right. (C) APB Northern blot run on the same 0Q and 100Q total RNA extracts. (D) Quantification of APB Northern blot by band densitometry.
主要实验及结论
该研究巧妙地利用了细菌tRNA鸟嘌呤转糖苷酶（TGT） 的催化选择性。这种酶能够将荧光团缀合的preQ₁类似物共价嵌入到tRNA中，但其先决条件是该tRNA尚未被Queuine修饰。换言之，这种标记技术直接反映了tRNA的欠修饰状态，将抽象的化学修饰差异转化为了直观的荧光信号。实验初期，研究人员在HEK293T和HT1080细胞系中进行了验证，通过APB北方印迹和变性凝胶成像证实，该方法具有极高的专一性和巨大的动态范围，标记信号在完全欠修饰的样本中极其显著，而在完全修饰的样本中则几乎消失。
Figure 2. Imaging relative queuine modification levels in 0Q and 100Q cells. (A) Schematic of the workflow to label queuine-unmodified tRNAs in cells for imaging of queuine modification. (B) Representative fluorescence microscopy images showing fixed 0Q and 100Q cells labeled with preQ1-Cy5 and TGT. (C) Quantification of fluorescence in 0Q and 100Q cells. Data represents individual fluorescence from three biological replicates (wells) from 5 individual images. (D) Raw flow cytometry data of 50,000 0Q and 100Q cells labeled with preQ1-Cy5 and StrandBrite Green. FL1 represents StrandBrite Green, and FL4 represents Cy5. (E) Normalized fluorescence intensity of 0Q and 100Q cell populations from flow cytometry analysis.
Figure 3. Imaging relative queuine modification levels in QTRT CRISPRi knockdown cells. (A) qPCR data showing expression of QTRT1 in HT1080 QTRT1 KD cells compared to the wild type. (B) Representative images showing fixed HT1080 WT and HT1080 QTRT1 KD cells labeled with preQ1-Cy5 and TGT. (C) Quantification of fluorescence in HT1080 WT and HT1080 QTRT1 KD cells. Data represents individual fluorescence from three biological replicates (wells) from 5 individual images.
随后，研究团队将该技术应用于固定细胞的共混成像。通过共聚焦显微镜观察发现，在缺乏Queuine的环境中生长的细胞，其荧光强度比富含Queuine环境下的细胞高出约3倍。为了进一步验证其生物学应用价值，研究人员利用CRISPRi技术敲低了人类TGT复合物的催化亚基QTRT1，同样观察到了显著的荧光增强，证明该技术能精准捕捉遗传因素导致的修饰水平波动。更具突破性的是，研究人员利用该方法监测了Queuine进入细胞后的动力学变化，发现在纤维肉瘤细胞系中的掺入速率明显慢于非癌上皮细胞。此外，通过与线粒体追踪染料共定位，研究惊人地发现线粒体tRNA的修饰速率慢于胞质tRNA，揭示了亚细胞层面Q修饰的不均一性，这是以往任何测序技术都无法观察到的空间细节。
Figure 4. Tracking kinetics of queuine incorporation into tRNAs with fluorescence imaging in HEK293T and HT1080 cells and queuine loss in HEK293T cells. (A) Representative fluorescence microscopy images showing images taken at various time points during treatment of queuine-starved HEK293T and HT1080 cells with 1 μM queuine. (B) Quantification of fluorescence from cells at each time point. Data represents individual fluorescence from three biological replicates (wells) from 5 individual images. (C) Representative fluorescence microscopy images showing images taken at various time points of queuine-treated 100Q cells switched into queuine-free media. (D) Quantification of fluorescence from cells at each time point. Data represents individual fluorescence from three biological replicates (wells) from 5 individual images.
Figure 5. Queuine modification occurs more slowly in the mitochondria than in the rest of the cytosol. (A) Colocalization of Cy5 signal with Mitotracker at early time points in a queuine incorporation kinetics experiment indicates that queuine is depleted in the mitochondria relative to the cytosol after treatment of queuine-starved cells with queuine. Cells were imaged on a confocal microscope using a 60x oil objective lens. (B) Pearson’s correlation coefficient of colocalization between Mitotracker and Cy5 signal was calculated at each time point for a representative set of images. Pearson’s coefficient was significantly higher at the 15-min and 30-min time points, indicating much greater colocalization at these time points.
总结及展望
这一研究不仅是首个能够以亚细胞分辨率成像tRNA Queuine修饰的技术，更为 epitranscriptomics（表观转录组学）的研究提供了一个通用的化学框架。通过将内源性RNA修饰状态转化为共价荧光信号，科学家现在可以在不破坏细胞结构的前提下，深入探索RNA修饰在疾病尤其是癌症中的动态演变。该方法的成功展示了酶促共价标记在解码生物大分子复杂化学状态方面的巨大潜力。未来，这种策略有望扩展到诸如m6A等其他重要的RNA修饰领域，为高通量遗传筛选和靶向表观转录组的药物研发开辟全新的道路。