Determination of tRNA aminoacylation levels by high-throughput sequencing.
Determination of tRNA aminoacylation levels by high-throughput sequencing.
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DOI：
10.1093/nar/gkx514
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发表时间：
2017-08-21
影响因子：
14.9
通讯作者：
Pan T
中科院分区：
文献类型：
--
作者：
Evans ME;Clark WC;Zheng G;Pan T
转移RNA（tRNA）在其3′端被氨基酸氨酰化（带电荷）时解码mRNA密码子。带电荷的tRNA在细胞中迅速翻转，并且已知带电荷的tRNA部分的变化是细胞对应激反应的有用参数。可以使用酸变性凝胶测量单个tRNA种类的tRNA充电分数，或者使用微阵列在基因组水平上进行比较。这些基于杂交的方法不能用于哺乳动物tRNA的高分辨率分析，由于它们的大的序列多样性。在这里，我们开发了一种高通量测序方法，能够以单碱基分辨率准确测定带电tRNA组分（Charged DM-tRNA-seq）。我们的方法利用了最近开发的DM-tRNA-seq方法，但包括额外的化学步骤，专门去除不带电荷的tRNA中的3′A残基。通过对相同测序反应中每个tRNA种类的A-末端读段与A+ C-末端读段的分数进行计数来获得充电分数。在HEK 293 T细胞中，大多数胞质tRNA的电荷水平>80%，而tRNASer和tRNAThr的电荷水平较低。使用酸变性凝胶验证这些低电荷水平。我们的方法应广泛适用于调查的tRNA充电作为一个参数在生物调节。
Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3′ end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3′A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNASer and tRNAThr are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation.
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DOI：
10.1073/pnas.1103698108
发表时间：
2011-10-04
影响因子：
11.1
作者：
Rudinger-Thirion, Joelle;Lescure, Alain;Frugier, Magali
通讯作者：
Frugier, Magali
影响因子：
4.1
作者：
Parisien, Marc;Wang, Xiaoyun;Pan, Tao
通讯作者：
Pan, Tao
影响因子：
48
作者：
Cozen AE;Quartley E;Holmes AD;Hrabeta-Robinson E;Phizicky EM;Lowe TM
通讯作者：
Lowe TM
影响因子：
7.7
作者：
Dittmar, KA;Sorensen, MA;Pan, T
通讯作者：
Pan, T
DOI：
10.1073/pnas.1211077110
发表时间：
2013-02-05
影响因子：
11.1
作者：
Subramaniam, Arvind R.;Pan, Tao;Cluzel, Philippe
通讯作者：
Cluzel, Philippe