Identification of the ARGONAUTE 1‐Enriched sRNomes and Targetomes by Integrated RIP‐Seq Data Analysis in Arabidopsis thaliana

In addition to microRNAs (miRNAs), some small interfering RNAs (siRNAs) such as trans-acting siRNAs are also associated with ARGONAUTE 1 (AGO1) for target cleavage in plants. Efficient identification of the AGO1-enriched small RNAs (sRNAs) and their targets became a challenging task. Here, we emphasized the utility of RIP (RNA immunoprecipitation) sequencing in probing the above issue. By analyzing the AGO1-RIP-sRNA-seq data, 42 991, 22 516, 34 555, and 19 754 AGO1-enriched sRNAs were identified from flowers, leaves, roots, and seedlings of Arabidopsis, respectively. RIP-RNA-seq of DCL1 and HYL1 enabled us to discover 14 miRNA precursor candidates encoding the AGO1-enriched sRNAs. Second, to reduce the false positive rate of small RNA binding site (SBS) predictions, we focused on the SBSs covered by the AGO1 RIP peaks representing the transcriptome-wide binding regions of this protein. Degradome-based cleavage site mapping enabled us to identify the targetomes of the AGO1-enriched sRNAs in distinct samples. Notably, a great portion of the sRNA-target pairs detected in the wild-type seedlings were absent in the ago1 mutants. The high AGO1 dependency demonstrated the reliability of our workflow for sRNA target identification. Moreover, GO (Gene Ontology) term enrichment analysis showed that the ecotype- and organ-specific targetomes were functionally related to environmental response and organ development, respectively. Finally, some of the AGO1-enriched sRNAs showed great potential of regulating specific long non-coding RNAs. Together, by employing RIP-seq, our study provided a promising workflow for identification of the AGO1-enriched sRNomes and their targetomes in plants.