Beyond microRNAs: Analysis of chimeric reads characterises the diverse targetome of AGO2-mediated regulation.

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 driver sequences, and identifies its targets primarily via a seed mediated partial complementarity process Despite numerous studies, a definitive experimental dataset of AGO2 driver-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 driver 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 drivers 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 driver class and experimentally validate several interactions. In conclusion, our comprehensive analysis of the AGO2 targetome broadens our understanding of its driver 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/ Contactpanagiotis.alexiou@um.edu.mt, stepanka.vanacova@ceitec.muni.cz