Understanding Piwi‐interacting RNA pathway in Drosophila The discovery of Piwi‐interacting RNA (piRNA) pathway in Drosophila Argonaute proteins are the core components in all known small RNA silencing pathways. Animal Argonaute proteins can be subdivided into AGO and PIWI clades based on the sequence similarities. piRNAs are a novel class of small RNAs, which were defined by their specific binding to the PIWI clade of Argonaute proteins—Piwi, Aubergine (Aub), and Argonaute3 (Ago3) in flies. The PIWI clade of Argonaute proteins and piRNAs are expressed only in the gonads where they play pivotal roles in repressing of transposons and maintaining genomic integrity during gametogenesis. Drosophila piRNAs were initially named as repeat associated small interfering RNAs (rasiRNAs) and thought to be a variant class of small interfering RNAs (siRNAs) produced and functioning through the RNA interference (RNAi) pathway. During my first year of graduate studies, we found that the production and function of rasiRNAs do not require the canonical machinery in RNAi pathway or microRNAs (miRNA) pathway. Instead, they are disproportionately antisense, specifically associated with Piwi and Aub, and rely their biogenesis and function on a unique set of genes including piwi, aub, spindle‐E, and armitage. Our data directly demonstrated that PIWI proteins and rasiRNAs compose a novel small RNA silencing pathway in Drosophila gonads. Shortly, piRNA pathways were discovered in zebrafish, mouse, rat, and human, suggesting conserved mechanisms in their biogenesis and function. Drosophila Hen1, methylates the 3´ ends of piRNAs We also found that the 3´ ends of Drosophila piRNAs are modified. To characterize the modification, I developed a sensitive method, which allowed us selectively label the modified nucleotides on the 3´ ends of piRNAs. Combining this method with the two‐dimensional thin layer chromatography analyses, we found that the 3´ ends of Drosophila piRNAs are 2´‐O‐methylated. This finding reminded us of the previous report on small RNA modification in plants, where RNA methyltransferase HEN1 catalyzes 2´‐O‐methylation on the 3´ ends of all small silencing RNAs. We found that the Drosophila homolog of HEN1 methylates the 3´ ends of piRNAs; unlike plant HEN1, Drosophila Hen1 specifically acts on single‐stranded RNA; in homozygous Drosophila hen1 mutants, both the length and abundance of piRNAs are decreased. These results suggested a possible function of the 2´‐O‐methylation by Hen1 in prevent piRNAs from degradation. Collapse of germline piRNAs in Ago3 null mutants reveals Drosophila somatic piRNA pathway Two subsequent studies cast light on the biogenesis of the 5´ ends of Drosophila piRNAs. These studies indicated that antisense piRNAs are disproportionately associated with Aub and Piwi, typically begin with a uracil, and appear to derive from the transcripts of large, transposon‐rich genomic loci, called piRNA clusters. Whereas sense piRNAs, while less abundant, are disproportionately bound to Ago3, and typically bear an adenosine at position 10. The prevailing ping‐pong model for piRNA biogenesis reflects the discovery that the first 10 nt of antisense piRNAs bound to Aub or Piwi are often complementary to the first 10 nt of sense piRNAs bound to Ago3. It suggests that the 5´ ends of antisense piRNAs bound to Aub or Piwi are defined by sense piRNAs bound to Ago3, and, reciprocally, that the 5´ ends of sense piRNAs bound to Ago3 are defined by antisense piRNAs bound to Aub or Piwi. At its core, the model proposes that piRNAs participate in an amplification loop in which transposon mRNAs trigger the production of new, antisense piRNAs. Ago3, guided by sense piRNAs, lies at the center of the amplification loop. We have isolated strong loss‐of‐function mutations in ago3, allowing the first genetic test for the ping‐pong model. Our data provides strong support for an amplification cycle in which Ago3 acts to amplify piRNA pools and to enforce on them a strong antisense bias, increasing the number of piRNAs that can act to destroy transposon mRNAs. Moreover, we found a second, somatic piRNA pathway in the follicle cells that surround the developing oocyte and that involves direct piRNA production and function through Piwi, without ping‐pong .