DescriptionTransfer RNA (tRNA) and ribosomal RNA (rRNA) are known for their textbook functions in the translation machinery. With further advances in the high-throughput sequencing, accumulating evidence has revealed their novel functionality as sources of short RNA fragments which may act as post-transcriptional regulators in various organisms. Such tRNA-derived fragments (tRFs) and rRNA-derived fragments (rRFs) have been shown to be implicated in many biological pathways, e.g., ageing, neuronal disorders and cancers, etc. Using large-scale computational analyses of various types of sequencing data, we characterized tRFs and rRFs in flies, mouse and human. We revealed the loading patterns of age-associated rRFs to different Argonaute proteins and suggested their roles in the ageing process of flies. We inferred the potential biogenesis pathway of human rRFs. By comprehensively analyzing the experimentally crosslinked target RNAs of tRFs and rRFs, we investigated the binding mechanisms of these molecules to their targets in the Argonaute proteins and suggested potential regulatory functions.