Ueno Daishin, Yamasaki Shotaro, Demura Taku, Kato Ko
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
J Biosci Bioeng. 2018 Jun;125(6):723-728. doi: 10.1016/j.jbiosc.2017.12.022. Epub 2018 Feb 1.
The major obstacle of efficient transgene expression seems to be gene silencing, and one of the important factors in gene silencing is mRNA stability. Regulation of mRNA stability is an important aspect of the control of gene expression. mRNAs are degraded by both exonucleolytic digestion and endonucleolytic cleavage. However, with the exception of small RNA-guided cleavage, the mechanisms underlying endonucleolytic cleavage-dependent RNA degradation remain to be elucidated. High-throughput approaches for genome-wide profiling of RNA cleavage sites, collectively termed degradome sequencing, have been developed by several groups. These analyses have contributed to the identification of mRNA cleavage sites in plants, but due to selection of poly (A) mRNA in library preparation, these approaches cannot identify cleavage sites in a fully accurate manner. To address this issue, we developed a new experimental method, truncated RNA end sequencing (TREseq), which enabled us to accurately identify many cleavage sites. TREseq can also be used to estimate the efficiency of mRNA cleavage, revealing differences in base frequencies near cleavage sites that reflect differences in cleavage efficiency. These results will contribute to gain important knowledge about the stability of the transgene mRNA in the future.
高效转基因表达的主要障碍似乎是基因沉默，而基因沉默的重要因素之一是mRNA稳定性。mRNA稳定性的调控是基因表达控制的一个重要方面。mRNA可通过核酸外切消化和核酸内切切割而降解。然而，除了小RNA引导的切割外，核酸内切切割依赖性RNA降解的潜在机制仍有待阐明。几个研究小组已经开发出了用于全基因组RNA切割位点分析的高通量方法，统称为降解组测序。这些分析有助于鉴定植物中的mRNA切割位点，但由于文库制备中对聚腺苷酸mRNA的选择，这些方法无法完全准确地鉴定切割位点。为了解决这个问题，我们开发了一种新的实验方法——截短RNA末端测序（TREseq），它使我们能够准确鉴定许多切割位点。TREseq还可用于估计mRNA切割效率，揭示切割位点附近碱基频率的差异，这些差异反映了切割效率的差异。这些结果将有助于未来获得有关转基因mRNA稳定性的重要知识。
