tRNA pool analysis of S. cerevisiae using tRNA-HySeq

For a typical RNA polymerase (pol) II transcribed budding yeast gene, the 5' -end is characterized by a nucleosome-free region (NFR) immediate upstream of the transcription start site (TSS), flanked by two well-positioned nucleosomes (-1 and +1) containing H2A.Z. A similar arrangement of nucleosomes containing H2A.Z is found on the genes transcribed by pol III, which reside in the NFR actively maintained by the chromatin remodeling complexes. We did genome-wide MNase-seq and ChIP-seq experiments to study the nucleosome arrangement near pol III transcribed genes. We also measured the levels of different tRNAs in the tRNA pool of the wild type and Spt16 mutant (*spt16-197*) cells using tRNA-HySeq method. Although, it is difficult to measure the primary transcripts of tRNA due to their quick processing and the sequence degeneracy of the tRNA isogenes; a comparison of the wild type and Spt16 mutant showed both increase or decrease of tRNA transcripts. The result suggest that Spt16 may not be necessary for the transcription per se of tRNA genes.针对典型的经RNA聚合酶II（RNA polymerase II）转录的出芽酵母基因，其5'端的特征为转录起始位点（transcription start site, TSS）上游紧邻无核小体区域（nucleosome-free region, NFR），侧翼带有两个携带组蛋白H2A.Z的定位良好的核小体（-1和+1核小体）。由RNA聚合酶III转录的基因也存在类似的核小体排布模式，这类基因的NFR由染色质重塑复合物主动维持。本研究开展了全基因组微球菌核酸酶测序（MNase-seq）与染色质免疫共沉淀测序（ChIP-seq）实验，以探究RNA聚合酶III转录基因附近的核小体排布情况。此外，我们采用tRNA-HySeq技术，检测了野生型与Spt16突变体（*spt16-197*）细胞的tRNA库中不同tRNA的表达水平。由于tRNA初级转录本会被快速加工，且tRNA同源基因存在序列简并性，因此难以对其进行定量检测；但对比野生型与Spt16突变体细胞的结果显示，tRNA转录本的丰度可出现上调或下调。上述结果表明，Spt16可能并非tRNA基因转录本身所必需的因子。