Comprehensive analysis of mRNA internal cleavage sites in Arabidopsis thaliana

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.