Evans Molly E, Clark Wesley C, Zheng Guanqun, Pan Tao
Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Nucleic Acids Res. 2017 Aug 21;45(14):e133. doi: 10.1093/nar/gkx514.
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.
转运RNA（tRNA）在其3'端被氨基酸氨酰化（负载）时对信使RNA（mRNA）密码子进行解码。负载的tRNA在细胞中快速周转，并且已知负载的tRNA组分的变化是细胞对应激反应的一个有用参数。可以使用酸性变性凝胶针对单个tRNA种类测量tRNA负载组分，或者在基因组水平上使用微阵列进行比较测量。由于哺乳动物tRNA具有较大的序列多样性，这些基于杂交的方法不能用于对其进行高分辨率分析。在此，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负载的tRNA组分（负载的DM-tRNA测序）。我们的方法利用了最近开发的DM-tRNA测序方法，但包括额外的化学步骤，专门去除未负载tRNA中的3'A残基。通过在同一测序反应中计算每个tRNA种类的以A结尾的读数与以A + C结尾的读数的比例来获得负载组分。在人胚肾293T细胞中，大多数胞质tRNA的负载水平> 80％，而丝氨酸tRNA和苏氨酸tRNA的负载水平较低。使用酸性变性凝胶验证了这些低负载水平。我们的方法应该广泛适用于将tRNA负载作为生物调节参数的研究。
