Argonaute proteins are instrumental in regulating RNA stability and translation. AGO2, the major mammalian Argonaute protein, is known to primarily associate with microRNAs, a family of small RNA 'guide' sequences, and identifies its targets primarily via a 'seed' mediated partial complementarity process. Despite numerous studies, a definitive experimental dataset of AGO2 'guide'-'target' interactions remains elusive. Our study employs two experimental methods-AGO2 CLASH and AGO2 eCLIP, to generate thousands of AGO2 target sites verified by chimeric reads. These chimeric reads contain both the AGO2 loaded small RNA 'guide' and the target sequence, providing a robust resource for modeling AGO2 binding preferences. Our novel analysis pipeline reveals thousands of AGO2 target sites driven by microRNAs and a significant number of AGO2 'guides' derived from fragments of other small RNAs such as tRNAs, YRNAs, snoRNAs, rRNAs, and more. We utilize convolutional neural networks to train machine learning models that accurately predict the binding potential for each 'guide' class and experimentally validate several interactions. In conclusion, our comprehensive analysis of the AGO2 targetome broadens our understanding of its 'guide' repertoire and potential function in development and disease. Moreover, we offer practical bioinformatic tools for future experiments and the prediction of AGO2 targets. All data and code from this study are freely available at https://github.com/ML-Bioinfo-CEITEC/HybriDetector/ .

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Centre for Molecular Medicine and Biobanking University of Malta Msida MSD 2080 Malta

Department of Applied Biomedical Science Faculty of Health Sciences University of Malta Msida MSD 2080 Malta

Faculty of Science National Centre for Biomolecular Research Masaryk University 62500 Brno Czech Republic

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