Comprehensive analysis of small RNA-seq data reveals that combination of miRNA with its isomiRs increase the accuracy of target prediction in Arabidopsis thaliana

Along with the canonical miRNA, distinct miRNA-like sequences called sibling miRNAs (sib-miRs) are generated from the same pre-miRNA. Among them, isomeric sequences featuring slight variations at the terminals, relative to the canonical miRNA, constitute a pool of isomeric sibling miRNAs (isomiRs). Despite the high prevalence of isomiRs in eukaryotes, their features and relevance remain elusive. In this study, we performed a comprehensive analysis of mature precursor miRNA (pre-miRNA) sequences from Arabidopsis to understand their features and regulatory targets. The influence of isomiR terminal heterogeneity in target binding was examined comprehensively. Our comprehensive analyses suggested a novel computational strategy that utilizes miRNA and its isomiRs to enhance the accuracy of their regulatory target prediction in Arabidopsis. A few targets are shared by several members of isomiRs; however, this phenomenon was not typical. Gene Ontology (GO) enrichment analysis showed that commonly targeted mRNAs were enriched for certain GO terms. Moreover, comparison of these commonly targeted genes with validated targets from published data demonstrated that the validated targets are bound by most isomiRs and not only the canonical miRNA. Furthermore, the biological role of isomiRs in target cleavage was supported by degradome data. Incorporating this finding, we predicted potential target genes of several miRNAs and confirmed them by experimental assays. This study proposes a novel strategy to improve the accuracy of predicting miRNA targets through combined use of miRNA with its isomiRs.除经典微RNA（canonical miRNA）外，同一前体微RNA（pre-miRNA）还可产生一类独特的类微RNA序列，即同源微RNA（sibling miRNAs，sib-miRs）。其中，与经典微RNA相比末端存在轻微序列变异的同源序列，构成了同源变异微RNA（isomeric sibling miRNAs，isomiRs）的序列库。尽管同源变异微RNA在真核生物中广泛存在，但其序列特征与生物学关联仍有待阐明。本研究针对拟南芥的成熟微RNA与前体微RNA（pre-miRNA）序列开展全面分析，以解析其序列特征与调控靶标，并全面探究了同源变异微RNA末端序列异质性对靶标结合的影响。综合分析结果表明，可通过整合微RNA与其同源变异微RNA构建全新的计算策略，以提升拟南芥中微RNA调控靶标的预测精度。少数靶标可被多个同源变异微RNA共同识别，但该现象并不普遍。基因本体（GO）富集分析显示，共同靶向的信使RNA（mRNA）在特定GO功能条目上存在富集。此外，将上述共同靶向基因与已发表文献中的验证靶标进行比对后发现，多数同源变异微RNA均可结合这些验证靶标，而非仅经典微RNA。降解组（degradome）数据进一步验证了同源变异微RNA在靶标剪切过程中的生物学功能。基于上述研究发现，本研究对多种微RNA的潜在靶标基因进行了预测，并通过实验手段验证了预测结果。本研究提出了一种通过整合微RNA与其同源变异微RNA，提升微RNA调控靶标预测精度的全新策略。