Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.

微小RNA（MicroRNAs, miRNAs）是一类长度约为21个核苷酸的小RNA（sRNAs），在植物中发挥着至关重要的调控作用。它们由具有茎环结构的前体经两次连续的类Dicer 1（DCL1）介导的切割反应生成。截至目前，科研人员已发现数百种植物miRNAs。然而，一个关键科学问题尚未得到解答：与miRNA前体反向互补（RC）的序列是否能够形成发夹状结构，并产生类似miRNA/miRNA*双链的小RNA双链？本研究基于小RNA高通量测序数据与二级结构预测方法，对16种植物中的该可能性展开了探究。最终在10种植物中鉴定出59条具备形成茎环结构、生成miRNA/miRNA*样双链潜力的反向互补序列，将其命名为反向互补miRNA前体（RC-miRNA前体）。与经典miRNAs不同，基于降解组数据，我们仅在拟南芥（Arabidopsis thaliana）和水稻（Oryza sativa）中鉴定到少量RC-miRNAs的切割靶点，而在大豆（Glycine max）中未发现任何靶点。令人意外的是，在拟南芥与水稻中，部分RC-miRNA靶点识别位点周围的基因组区域呈现出特异性DNA甲基化修饰。综上，本研究报道了一类全新的miRNA——反向互补miRNAs（RC-miRNAs），其由miRNA前体的反义链生成。基于研究结果，我们推测：成熟RC-miRNAs可能通过靶点切割发挥微弱的调控活性，同时可能通过引导序列特异性DNA甲基化，展现出类似小干扰RNA的功能活性。