De novo origination of MIRNAs through generation of short inverted repeats in target genes
MIRNA (MIR) gene origin and early evolutionary processes, such as hairpin precursor sequence origination, promoter activity acquirement and the sequence of these two processes, are fundamental and fascinating subjects. Three models, including inverted gene duplication, spontaneous evolution and transposon transposition, have been proposed for de novo origination of hairpin precursor sequence. However, these models still open to discussion. In addition, de novo origination of MIR gene promoters has not been well investigated. Here, I systematically investigated the origin of evolutionarily young polyphenol oxidase gene (PPO)-targeting MIRs, including MIR1444, MIR058 and MIR12112, and a genomic region termed AasPPO-as-hp, which contained a hairpin-forming sequence. I found that MIR058 precursors and the hairpin-forming sequence of AasPPO-as-hp originated in an ancient PPO gene through forming short inverted repeats. Palindromic-like sequences and imperfect inverted repeats in the ancient PPO gene contributed to initiate the generation of short inverted repeats probably by causing errors during DNA duplication. Analysis of MIR058 and AasPPO-as-hp promoters showed that they originated in the 3ʹ-flanking region of the ancient PPO gene. Promoter activities were gained by insertion of a CAAT-box and multiple-copper-response element (CuRE)-containing miniature inverted-repeat transposable element (MITE) in the upstream of AT-rich TATA-box-like sequence. Gain of promoter activities occurred before hairpin-forming sequence origination. Sequence comparison of MIR1444, MIR058 and MIR12112 promoters showed frequent birth and death of CuREs, indicating copper could be vital for the origination and evolution of PPO-targeting MIRs. Based on the evidence obtained, a novel model for plant MIR origination and evolution is proposed.